Multiple morphogenic culture systems cause loss of resistance to cassava mosaic disease

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1 Chuhn et l. BMC Plnt Biology (8) 8: RESEARCH ARTICLE Open Aess Multiple morphogeni ulture systems use loss of resistne to ssv mosi isese Rj Deepik Chuhn, Getu Beyene n Nigel J. Tylor * Astrt Bkgroun: Morphogeni ulture systems re entrl to rop improvement progrms tht utilize trnsgeni n genome eiting tehnologies. We previously reporte tht CMD-type ssv (Mnihot esulent) ultivrs lose resistne to ssv mosi isese (CMD) when psse through somti emryogenesis. As result, these plnts nnot e evelope s prouts for eployment where CMD is enemi suh s su-shrn Afri or the Inin su-ontinent. Result: In orer to inrese unerstning of this phenomenon, Afrin ssv ultivrs were sreene for resistne to CMD fter regenertion through somti emryogenesis. Fifteen ultivrs were shown to retin resistne to CMD through somti emryogenesis, onfirming tht the existing trnsformtion n gene eiting systems n e employe in these geneti kgrouns without ompromising resistne to geminivirus infetion. CMD-type ultivrs were lso sujete to plnt regenertion vi ulogenesis n meristem tip ulture, resulting in 5 6% n 5 % of regenerte plnt lines losing resistne to CMD respetively. Conlusions: This stuy provies ler eviene tht multiple morphogeni systems n result in loss of resistne to CMD, n tht somti emryogenesis per se is not the unerlying use of this phenomenon. The informtion esrie here is ritil for interpreting genomi, trnsriptomi n epigenomi tsets ime t unerstning CMD resistne mehnisms in ssv. Keywors: Cssv, Cssv mosi isese, Meristem tip ulture, Orgnogenesis, Somti emryogenesis Bkgroun Cssv mosi isese (CMD) is enemi throughout Su-Shrn Afri n the Inin su-ontinent. Effetive resistne to the whitefly-vetore geminiviruses tht use CMD is essentil to seure yiels for ssv frmers ross these regions. Three geneti soures of CMD resistne, i.e. CMD, CMD n CMD, hve een ientifie. CMD resistne ws introgresse from Mnihot glziovii n unerstoo to e multigeni n reessive, while CMD is monolous, ominnt in nture n ws ientifie in lnres ollete in Nigeri n Benin/Togo [, ]. CMD rries the CMD lous plus n itionl QTL []. In ll three resistne types, the unerlying genes n moleulr mehnisms remin unknown. We reently reporte tht ll plnts of * Corresponene: ntylor@nforthenter.org Donl Dnforth Plnt Siene Center, St. Louis, MO, USA CMD-type ultivrs regenerte through somti emryogenesis lose resistne to CMD n evelop severe mosi symptoms when inoulte with infetious geminivirus lones in the greenhouse, n when expose to viliferous whiteflies in the fiel. Cultivrs teste tht rry CMD n CMD resistne mehnisms i not suffer from this phenomenon with plnts regenerte through somti emryogenesis remining resistnt to CMD [4]. Uniform n onsistent loss of mjor trit suh s virus resistne in multiple ultivrs y simple pssge through emryogenesis is unique in the literture. Inresing unerstning of why CMD resistne is ompromise in this mnner is impertive to the suess of ssv enhnement progrms. In generl, phenotypi vritions in plnts reovere through tissue ulture n e ttriute to geneti or epigeneti hnges. Chnges in the DNA methyltion sttus of the ssv genome The Author(s). 8 Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( pplies to the t me ville in this rtile, unless otherwise stte.

2 Chuhn et l. BMC Plnt Biology (8) 8: Pge of were reporte in plnts regenerte vi meristem tip ulture y Kitimu et l. [5]. The single lous, ominnt nture of CMD mkes it highly fvore y reeers s soure of resistne to generte improve plnting mterils [6]. Reline on single gene mehnism, however, risks evolution of the pthogen to overome the resistne. Inee, rekown of CMD-meite resistne ws reporte reently uner greenhouse onitions y Nunguru, et l. [7]. Avne iotehnologies in ssv rely on inution of somti emryogenesis to generte the totipotent tissues utilize for trnsgene integrtion n elivery of gene eiting regents [8, 9]. Geneti moifition in this mnner must e hieve without losing resistne to CMD, trit tht is essentil in ll enhne ssv germplsm intene for eployment in Afri n Ini. We report here further eviene for loss of funtionl CMD-meite resistne when tissues re psse through morphogeni ulture systems. In ition to somti emryogenesis, informtion is presente esriing the effets of ulogenesis n meristem tip ulture on loss of resistne to CMD in regenerte plnts. Methos Mei omposition n ulture onitions Compositions of ulture mei use in this stuy followe Chuhn, et l. [] for inution of orgnize emryogeni strutures (OES) n frile emryogeni llus (FEC); Chuhn n Tylor [] for orgnogenesis; n the Interntionl Institute of Tropil Agriulture Hnook [] for meristem tip ulture. Mei omponents, ntiiotis, growth regultors n itives were proure from Sigm (St. Louis, MO, USA). Met-topolin (mt) use for regenertion of plnts through orgnogenesis ws otine from Duhef Biohemie, The Netherlns. All in vitro ultures were inute t 8 ± o Cwith6h light/ 8 h rk photoperio uner fluoresent lmps t 75 μmol m s - unless otherwise speifie. Plnt mteril n gene onstruts In vitro shoot ultures of CMD-type ssv ultivr TMS 57, CMD-type ultivrs TME 49, TME B7, CMD-type ultivrs TMS 98/58, TMS 98/55, TMS 96/6 n other ultivrs with unknown CMD-types NR /55, TMS 98/, TMS /4, TMS 9/ 57, TMS /6, TMS 9/4, TMS 98/, TMS 9/6, TMS /7, TMS 95/89 n 6444 were otine from IITA, Nigeri (Tle ). Stem uttings of CMD-type ultivrs NASE, NASE 4 n CMD-type ultivrs TME 4, TME 4 were importe from the Ntionl Crops Resoures Reserh Institute (NCRRI), Ugn, n TME 7 from IITA ollete from frmer fiels in Nigeri. Stem uttings were estlishe uner in vitro onitions t the Donl Dnforth Plnt Siene Center (DDPSC), St. Louis, MO, USA. Axillry us tht evelope from the stems were exise n estlishe in tissue ulture following methos esrie y Tylor, et l. [] n Chuhn, et l. []. CMD suseptile plnts of TME 4 were otine y regenertion from frile emryogeni llus (FEC-TME 4) []; [4] n serve s known negtive ontrols for greenhouse trils. Agroterium tumefiens strin LBA444 hroring pcambia-se inry vetor ontining the enhne green fluoresent protein gene (egfp) uner ontrol of the Culiflower mosi virus (CMV) 5S promoter ws use for trnsformtion experiments, following proeures esrie y Chuhn, et l. []. Proution of orgnize emryogeni strutures (OES) n plnt regenertion Inution of orgnize emryogeni strutures (OES) ws performe s esrie y Tylor, et l. [] n Chuhn, et l. []. Immture lef loe explnts were exise from 4- to 6-week-ol miropropgte shoot ultures n ple on DKW/Juglns sl slts [4] (PhytoTehnology Lortories, Knss, USA) plus Murshige n Skoog (MS) [5] vitmins, supplemente with % w/v surose n 5 μm pilorm (DKW 5P). Cultures were inute in the rk t 8 C for 4 weeks. Eight lef loe explnts were ulture per plte with five pltes per ultivr, n experiments replite three times. The numer of explnts forming OES ws ssesse 5 weeks fter explnting. Plnts were regenerte 8 weeks fter lef loe explnt initition y exising OES from the non-emryogeni tissues n suulture onto MS mei ontining % surose w/v (MS) n μm mt soliifie with.% w/v gelzn []. Between eight n olonies of OES were ulture in eh plte. After 4 weeks, iniviul otyleon stge emryos were seprte from eh other n suulture onto fresh mei of the sme type. Germinting shoots possessing two to three true leves were trnsferre for rooting to MS mei supplemente with % w/v surose n soliifie with.8% w/ v Nole gr. Agroterium-meite trnsformtion n plnt regenertion Frile emryogeni llus (FEC) proue from six ultivrs TMS 98/55, TMS /4, TMS /6, TMS 9/4, TME B7 n TME 49 ws trnsforme with Agroterium tumefiens strin LBA444 hroring pcambia-se inry vetor rrying egfp following the metho esrie y Chuhn, et l. []. Agroterium suspension t n OD 6 of.5 ws use to inoulte FEC n the ultures were kept t C uner onstnt light. Three to 4 ys fter the inoultion, the

3 Chuhn et l. BMC Plnt Biology (8) 8: Pge of Tle Inution of orgnize emryogeni strutures (OES), frile emryogeni strutures (FEC) from ssv ultivrs n response to MeSPY-VIGS ssv mosi isese hllenge Cultivr nme Resistne type Orgnize emryogeni strutures (OES) inution frequeny (%) Frile Emryo-geni Cllus (FEC) inution (Yes/No) Numer of e plnts/totl plnts hllenge with MeSPY-VIGS Resistne/ suseptiility to ssv mosi isese Wiltype OES-erive Wiltype OES-erive NASE CMD 4 No /8 /8 Resistnt Resistnt NASE 4 CMD 8 Yes /9 / Resistnt Resistnt TMS 57 CMD 8 No /9 /8 Resistnt Resistnt TME 4 CMD 8 Yes /9 7/7 Resistnt Suseptile TME B7 CMD 95 Yes /9 6/6 Resistnt Suseptile TME 49 CMD 58 Yes 7/9 6/6 Suseptile Suseptile TMS 96/6 CMD 6 No /6 /9 Resistnt Resistnt TMS 98/55 CMD 55 Yes /8 /9 Resistnt Resistnt TMS 98/58 CMD 66 No /7 / Resistnt Resistnt TMS 9/6 Unknown 89 Yes /8 /7 Resistnt Resistnt TMS 9/57 Unknown 76 No / /5 Resistnt Resistnt TMS 95/89 Unknown No / NA Resistnt Not teste TMS 98/ Unknown 79 No /6 /4 Resistnt Resistnt NR/55 Unknown 5 No /9 /8 Resistnt Resistnt TMS 9/4 Unknown 5 Yes / /9 Resistnt Resistnt TMS 98/ Unknown 78 No / / Resistnt Resistnt TMS /4 Unknown 59 Yes / /9 Resistnt Resistnt TMS /6 Unknown 66 Yes /7 /8 Resistnt Resistnt TMS /7 Unknown 64 No /8 /9 Resistnt Resistnt Munmli Unknown Not etermine Not teste / 5/5 Suseptile Suseptile 6444 Suseptile 9 Yes 9/9 NA Suseptile Not teste FEC erive TME 4 use s ontrol (FEC-TME 4) Agroterium ws wshe off from the FEC. The tissues were then selete on mei ontining 7.5 μm promomyin followe y trnsfer to emryo mturtion mei ontining 45 μm promomyin. The otyleon stge emryos were germinte n roote on seletion free mei. GFP-expressing tissues were visulize uner Nikon C54 isseting mirosope equippe with n exittion filter of 46 5 nm n rrier filter, 5 LP t ifferent stges fter trnsformtion n sore s esrie y Chuhn, et l. []. Three replites were estlishe per ultivr for eh tretment n trnsformtion experiments repete two times. Non-trnsgeni plnts for use s negtive ontrols were reovere from non-trnsforme FEC. Regenertion of ssv plnts through orgnogenesis Plnts of CMD-type ultivrs TME 7 n TME 4 were regenerte from lef-petiole explnts following Chuhn n Tylor []. Lef-petiole explnts were exise from mother plnts pre-trete with μm mt for 4 weeks, ulture on MS meium supplemente with % w/v surose, μm,4-d n μm mt for 7 ys, followe y trnsfer to MS meium ontining 6 μm mt. Tissues were suulture onto fresh mei of the sme type every weeks. Regenerte shoots. to.5 m in length were trnsferre to MS mei for rooting n plntlet estlishment. Regenertion of ssv plnts through meristem tip ulture Plnts of CMD-type ultivrs TME 7, TME 4, TME 4, CMD-type ultivr TMS 75 n CMD-type ultivr TMS 98/55 were regenerte through meristem tip ulture following the metho esrie y IITA []. Six- to eight-week-ol in vitro miropropgte mother plnts ulture on MS mei supplemente with % w/v surose (MS) n soliifie with.8% w/v nole gr were use s the explnt soure. Lef primori were remove from the shoot tip using hypoermi neele uner stereomirosope (Olympus SMZ5) until the meristemti ome ws visile. The meristem tip (~.5 mm in size) ws exise n ple on MS

4 Chuhn et l. BMC Plnt Biology (8) 8: Pge 4 of sl mei supplemente with. g/l inositol,.8 g/l enine sulfte,.7 μm NAA (-npthlene eti i),. μm BAP (6-enzylminopurine),. μm GA (gierelli i), % w/v surose n soliifie with.4% w/v Nole gr. Cultures were inute in the rk for two to 4 weeks t 8 ± o C. Regenerting shoots were roote on MS mei. Between 8 n 4 meristem tip explnts were exise n ulture for eh ultivr with the numer of explnts inuing shoots suitle for trnsferring to rooting mei ssesse fter 5 weeks in ulture. Inoultion of the plnts with geminiviruses in the greenhouse Plnts tht reovere through ll morphogeni pthwys were propgte long with the ontrols on MS mei n soliifie with.% w/v gelzn. After three to 4 weeks of ulture, plntlets were trnsferre to Ffr 5 growing mixture in -inh pots n ple on mist enh t % reltive humiity for 7 ys followe y trnsfer to the open enh t 8 ± C y/ 5 ± C night temperture in 4 h light/ h rk photoperio t 8 to 4 μmolm s - irrine n 8 9% reltive humiity n llowe to grow for weeks []. Plnts 8 to 9 m in height were trnsferre to greenhouse n grown t o C y/ 7 C night yle with 7 95% reltive humiity. A rpi VIGS-se sreening metho evelope y Beyene et l. [6] ws employe to etermine the CMD sttus of the plnts reovere from OES n meristem tip ulture. Four- to six-week-ol plnts were inoulte with plsmi DNA of MeSPY (Mnihot esulent SPY) -VIGS n the DNA-B omponent of Est Afrin ssv mosi virus (EACMV-K) using Helios Gene Gun (BioR, Herules, Cliforni). This uses silening of MeSPY whih les to shoot-tip nerosis n eth of the plnt in CMD-suseptile ssv plnts within -4 weeks of inoultion wheres the CMD-resistnt plnts remin helthy. The shoot-tip nerosis n eth of plnts were sore ommening 4 ys fter inoultion. Plnts reovere from FEC, meristem tip ulture n orgnogenesis were inoulte with ssv geminiviruses following Beyene, et l. [4]. Four-week-ol greenhouse-grown plnts were inoulte with infetious lones of Est Afrin ssv mosi virus (EACMV-K) DNA-A GenBnk: AJ7754 n DNA-B GenBnk: AJ7495) [7, 8] n Afrin ssv mosi virus Cmeroon strin (ACMV-CM) DNA-A GenBnk AF5 n DNA-B GenBnk AF5 [9] using Helios Gene Gun (BioR, Herules, CA, USA). Inoulte plnts were ssesse for CMD symptoms strting 7 ys post inoultion (DPI), with symptom severity sore on sle of 5 [] twie per week. Results Sreening ssv ultivrs for CMD resistne fter pssge through somti emryogenesis We previously reporte tht CMD-type ssv plnts tht h een regenerte through somti emryogenesis lose resistne to CMD ut tht no suh effet is oserve in ultivrs rrying CMD n CMD resistne mehnisms [4]. To investigte this phenomenon further, ssv ultivrs (Tle ) from Est n West Afri were psse through somti emryogenesis y inuing OES from lef explnts [, ]. Plnts regenerte from OES were hllenge with n infetious VIGS lone of EACMV-K moifie to rry sequenes tht trget MeSPY. Plnts with funtionl resistne to geminviruses reover from this inoultion, while shoot-tip of suseptile plnts wilt n ie within two to 4 weeks fter inoultion [6]. Plnts were lso inoulte with the infetious lone of EACMV- K [4]. Similr results were otine from oth CMD hllenge methos. All ultivrs teste unerwent somti emryogenesis to proue OES, with effiienies vrying from s high s 9% in 6444, to only % in TMS 95/89 (Tle ). Plnts were regenerte for ll ultivrs (exept TMS 95/89), estlishe in the greenhouse n sujete to inoultion with MeSPY-VIGS. Wil-type plnts of the known CMD-types TME 4 n TME 7 emonstrte resistne to CMD n survive the MeSPY-VIGS hllenge. Conversely, shoot-tips of plnts of CMD-type ultivrs regenerte from OES strte to wilt 4 DPI n susequently ie (Fig. ). As onsistently oserve in our lortory, wil-type plnts of the CMD-type ultivr TME 49 possess low-level resistne to infetion with the infetious lone EACMV-K, lthough it oes possess roust resistne to ACMV (t not shown). Wil-type plnts of ssv ultivrs Munmli n 6444 re CMD suseptile n remine so fter regenertion through somti emryogenesis. The remining 5 ultivrs, whether rrying CMD, CMD or unknown types of resistne to CMD, remine fully resistnt to inoultion with MeSPY-VIGS fter pssge through somti emryogenesis (Tle ). FEC is the preferre trget tissue for geneti trnsformtion n is eing pte for the pplition of gene eiting in ssv [8 ]. OES from ll ultivrs shown in Tle were suulture onto Gresshoff n Doy [] -se meium in orer to proue FEC. FEC ws suessfully generte from ultivrs, inluing six West Afrin vrieties tht hve not een reporte previously (Tle ). Trnsgeni plnt proution ws ttempte y Agroterium-meite trnsformtion of FEC in the six ultivrs TMS 98/55, TMS /4, TMS /6, TMS 9/4, TME B7 n TME 49.

5 Chuhn et l. BMC Plnt Biology (8) 8: Pge 5 of Fig. Response of wil-type (left) n orgnize emryogeni strutures (right) erive plnts to inoultion with MeSPY-VIGS to etermine resistne to ssv mosi isese. Silening of MeSPY using MeSPY-VIGS les to shoot-tip nerosis n eth of CMD suseptile ssv plnts within 4 weeks fter inoultion. TME B7. TMS 98/. NASE 4. Munmli GFP-expressing plnt lines of TMS 98/55 (Fig. 4 & ) were estlishe in the greenhouse n inoulte with EACMV-K (Fig. ). Of four TMS 9/ 4 FEC-erive, five TMS 98/55 FEC-erive, n 4 trnsgeni GFP-expressing TMS 98/55 inepenent lines hllenge, ll plnts reovere to isply no mosi symptoms within five to 6 weeks fter hllenge (Fig. 4). This t inites tht resistne to CMD ws retine through ll stges of GFP-expressing llus lines were reovere in ll ses (Figs. n ). As esrie previously [], trnsformtion ws signifintly more effiient if moxltm ws inlue in the ulture meium prior to o-ulture with Agroterium (Fig. ). Trnsgeni plnts were reovere from ultivrs TMS 98/55, TMS /6 n TMS 9/4, in ition to TME 49 n TME B7. FEC-erive plnts of TMS 9/4 (Fig. 4 & ) n TMS 98/55 n trnsgeni e WT WT- TME 4 FEC- TME 4 WT- TMS 98/55 GFP- TMS 98/55 Fig. Agroterium-meite geneti trnsformtion of TMS 98/55 n response of trnsgeni plnts to inoultion with the infetious geminivirus lone EACMV-K. trnsient GFP expression fter 4 ys o-ulture with A. tumefiens. GFP-expressing llus line. GFP-expressing somti emryos on regenertion mei. Trnsgeni roote plnt. e Response of trnsgeni n miropropgte wil-type plnts to EACMV-K t ys post inoultion

6 Chuhn et l. BMC Plnt Biology (8) 8: Pge 6 of Av. numer of stle GFP iviing llus lines per SCV numer of roote events reovere per SCV Av No moxltm 5 mg/l moxltm TMS 98/55 TMS /4 TMS /6 TMS 9/4 TME B7 TME 49 No moxltm 5 mg/l moxltm Cssv ultivrs TMS 98/55 TMS /4 TMS /6 TMS 9/4 TME B7 TME 49 Cssv ultivrs Fig. Stle GFP-expressing trnsgeni events reovere from frile emryogeni llus (FEC) of ifferent ssv ultivrs. Averge numer of GFP positive llus lines otine fter 5 weeks of o-ulture. Averge numer of GFP positive roote events otine fter 4 5 months of o-ulture. Vlues re Averge ± SE, Numer of experiments one = n Replitions = per experiment somti emryogenesis (OES n FEC), geneti trnsformtion n plnt regenertion (Fig. e). Effet of orgnogenesis n meristem tip ulture on CMD resistne We reently esrie novel regenertion system in ssv y whih plnts re reovere from ifferent explnt types vi ulogenesis. Explnts re first ulture on meium ontining μm,4-d n μm mt for 7 ys, followe y suulture onto meium supplemente with 6 μm mt []. Shoots tht regenerte on the seon-stge meium originte from hr, rk green olore llus, with no eviene for the ourrene of somti emryogenesis. Plnts of CMD-type ultivrs TME 4 n TME 7 were regenerte from lef-petiole explnts ulture on mt [], estlishe in the greenhouse n inoulte with MeSPY-VIGS n EACMV-K to etermine if they h retine resistne to CMD. Loss of resistne to CMD ourre in oth ultivrs, ut only from portion of the regenerte plnt lines. In TME 7, six out of plnt lines regenerte through ulogenesis h lost resistne to CMD (Fig. 5 & ; Tle ). Of inepenent TME 4 regenernt lines inoulte with MeSPY-VIGS, seven lines were foun to hve retine resistne, n four to hve eome suseptile to CMD (Fig. 5 & ; Tle ). All lonl replites erive from given regenerte plnt line ehve in the sme mnner, whether resistnt or suseptile. When hllenge with the EACMV infetious lone ECAMV-K, the sme plnt lines from oth ultivrs remine resistnt or suseptile s ssesse y their ility to reover from CMD symptoms (Aitionl file : Figure S). Meristem tip ulture is well-estlishe metho for reovering pthogen-free plnts in ssv n mny other plnt speies []. The CMD-type ultivrs TME 4, TME 7 n TME 4, the CMD-type ultivr TMS 75 n CMD-type TMS 98/55 were sujete to meristem tip ulture to etermine effets of this tissue ulture system on CMD resistne (Tle ). Mximum plnt regenertion ws oserve in TME 4 followe y TME 7 n TMS 98/55. TME 4 showe the lowest shoot regenertion rte with only % explnts inuing shoots. When inoulte with MeSPY-VIGS, two out of 7 regenerte plnt lines in TME 7 n one out of 9 regenernts in TME 4 were foun to hve eome

7 Chuhn et l. BMC Plnt Biology (8) 8: Pge 7 of WT- TME 4 WT- TMS 9/4 WT FEC-TMS 9/4 FEC-TME 4 WT-TME 4 WT-TMS 98/55 WT-6444 GFP-TMS 98/55 FEC- TME 4 (%) plnts CMD symptomti Av Av. CMD symptomti plnts (%) WT- TME 4 WT- TMS 9/4 WT FEC-TMS 9/4 FEC-TME 4 5 WT-TME 4 WT-TMS 98/55 WT-6444 GFP-TMS 98/55 FEC- TME 4 Av. CMD severity sore ( -5) 4 Av. CMD severity sore (-5) Fig. 4 Response of non-trnsgeni n trnsgeni ssv plnts to inoultion with the infetious geminivirus lone EACMV-K. Nontrnsgeni n trnsgeni plnts of TMS 9/4 n CMD-type ultivr TMS 98/55, respetively, were generte from FEC. Perentge of ssv mosi isese (CMD) symptomti plnts of FEC-erive n miropropgte TMS 9/4. Averge CMD symptom severity sores (sle 5) on FEC-erive n miropropgte TMS 9/4. Perentge of CMD symptomti plnts of trnsgeni GFP expressing TMS 98/ 55 n wil-type TMS 98/55. Averge CMD symptom severity sores (sle 5) on GFP-expressing TMS 98/55 n wil-type TMS 98/ 55. Plnt stems were ut k 48 ys fter iolisti inoultion n CMD ssesse on new lef growth. Breks in the x xis inite lpse in shoot regrowth fter stem ut-k suseptile to CMD. The remining plnt lines in these n the other ultivrs teste retine resistne to CMD, reovering to estlish helthy plnts (Tle, Fig.6). Similr results were otine when the selete meristem tip-erive plnts were hllenge with the reltively mil infetious lone ACMV-CM (Fig. 7). Disussion Morphogeni ulture systems re entrl to the proution of trnsgeni ssv plnts n re eing pte for gene eiting pplitions [8, 9]. In mny ses, the intention is to eploy the resulting enhne mterils to frmers n/or reeers. Compromise resistne to CMD within suh plnt lines is therefore signifint onern. Beyene et l. [4] reporte tht ssv ultivrs possessing the ominnt, monolous CMD-type resistne lost resistne to CMD when psse through somti emryogenesis. It is essentil tht full unerstning of the evelopmentl n moleulr mehnisms unerlying loss of resistne to CMD is eluite. This is require to seure long-term onfiene in ssv plnts regenerte through tissue ulture, to enle improvement of CMD-type ultivrs through geneti engineering n gene-eiting tehnologies, n to unerstn if n how morphogeni systems oul lso result in loss of ritil trits in other rops. The ojetives of the present stuy were to inrese unerstning of this phenomenon y sreening wier popultion of West Afrin elite ssv ultivrs for resistne to CMD fter somti emryogenesis, n to etermine if lterntive morphogeni systems lso result in loss of CMD resistne. Tle Response of orgnogenesis-erive plnts to MeSPY-VIGS hllenge Cultivr nme No. of e inepenent regenernts/totl regenernts hllenge with MeSPY-VIGS Perentge CMD suseptile plnts TME 7 6/ 7 TME 4 4/ 6

8 Chuhn et l. BMC Plnt Biology (8) 8: Pge 8 of (%) plnts CMD symptomti Av WT- TME 7 FEC- TME 7 ORG- TME 7 (resistnt) ORG- TME 7 (suseptile) Av. CMD symptomti plnts (%) WT- TME 4 FEC- TME 4 ORG- TME 4 (resistnt) ORG- TME 4 (suseptile) ( to 5) sore severity Ave. 5 4 WT- TME 7 FEC- TME 7 ORG- TME 7 (resistnt) ORG- TME 7 (suseptile) Ave. severity sore ( to 5) 5 4 WT- TME 4 FEC- TME 4 ORG- TME 4 (resistnt) ORG- TME 4 (suseptile) Fig. 5 Response of orgnogenesis-erive plnts to inoultion with n infetious geminivirus lone EACMV-K. Perentge of CMD symptomti plnts of orgnogenesis-erive (ORG-TME 7) n wil-type CMD-type ultivr TME 7. Averge CMD symptom severity sores (sle 5) on orgnogenesis-erive n wil-type TME 7. Perentge of CMD symptomti plnts of orgnogenesis-erive (ORG-TME 4) n wil-type CMD-type ultivr TME 4. Averge CMD symptom severity sores (sle 5) on orgnogenesis-erive n wil-type plnts of TME 4. Plnt stems were ut k t 48 ys fter iolisti inoultion n CMD ws ssesse on new lef growth. Breks in the x xis inite lpse in shoot regrowth fter ut-k. n = 6 for ORG-TME 7 (resistnt), n = 6 for ORG-TME 7 (suseptile), n = 7 for ORG-TME 4 (resistnt), n = 4 for ORG-TME 4 (suseptile) Twenty-one ssv ultivrs were psse through somti emryogenesis n sujete to CMD hllenge uner greenhouse onitions. While CMD-type ssv eme suseptile in the mnner reporte y Beyene, et l. [4], 5 elite ssv ultivrs were onfirme to retin resistne to CMD when regenerte from somti emryos. FEC proue from TMS 98/55, TMS / 4, TMS /6 n TMS 9/4 ws foun to e menle to Agroterium-meite trnsformtion. In ll ses, use of moxoltm signifintly enhne proution of trnsgeni tissues n plnts. Roust resistne, equivlent to tht of the non-moifie wil-type plnts, ws emonstrte in ultivrs TMS 98/55 n TMS 9/4 fter regenertion from ll stges of somti emryogenesis n in trnsgeni plnts of TMS 98/ 55. High onfiene n e ple, therefore, on the use of existing somti emryogenesis protools to introue esirle trits through trnsgeni or gene eiting tehnologies in these ultivrs. CMD-type ultivrs re wiely grown y frmers in Est, West n Centrl Afri n employe in reeing progrms [, 6, ]. There is esire to pply iotehnology to improve these vrieties for trits inluing resistne to CBSD [4 6], nutritionl enhnement [7, 8] Tle Response of meristem tip-erive plnts to inoultion with MeSPY -VIGS hllenge Cultivr nme No. of explnts (meristem tip) No. of explnts forming shoots Perentge shoot regenertion No. of e inepenent regenernts/totl regenernts Perentge CMD suseptile plnts estlishe hllenge with MeSPY-VIGS TME /7 TME /9 5 TME / TMS / TMS 98/55 58 / Explnts were setup in two seprte experiments

9 Chuhn et l. BMC Plnt Biology (8) 8: Pge 9 of e f Fig. 6 Response of meristem tip ulture-erive plnts of TME 7, TME 4, TME 7, TMS 98/55 to inoultion with MeSPY-VIGS. Silening of MeSPY using MeSPY-VIGS les to shoot-tip nerosis n eth of the of CMD suseptile ssv plnts within 4 weeks. CMD resistnt miropropgte TME 7 plnt. CMD suseptile meristem tip-erive TME 7 plnt. CMD resistnt meristem tip-erive TME 7 plnt. CMD resistnt wil-type TME 4 plnt. e CMD suseptile meristem tip-erive TME 4 plnt. f CMD resistnt meristem tip-erive TME 4 plnt Av. CMD symptomti plnts (%) FEC- TME 4 MTC-TME 7-6 MTC-TME 7- MTC-TME WT-TME 4 MTC-TME 4- MTC-TME Av. CMD symptomti plnts (%) WT-TME 7 MTC-TME 7- MTC-TME 7- WT-TME 7 MTC-TME 7- MTC-TME FEC- TME 4 MTC-TME 7-6 MTC-TME 7- MTC-TME 7-7 WT-TME 4 MTC-TME 4- MTC-TME 4-8 FEC- TME 4 MTC-TME 4-9 MTC-TME4- MTC-TME 4- Av. CMD severity sore (-5) Av. CMD severity sore (-5) MTC-TME 4- MTC-TME FEC- TME 4 MTC-TME Fig. 7 Response of meristem tip-erive plnts of ssv to inoultion with infetious geminivirus lone ACMV-CM. Perentge of CMD symptomti plnts of meristem tip-erive CMD-type ultivr TME 7 plnts. Averge CMD symptom severity sores (sle 5) on meristem tip-erive TME 7 plnts. Perentge of CMD symptomti plnts of meristem tip-erive CMD-type ultivr TME 4 plnts. Averge CMD symptom severity sores (sle 5) on meristem tip-erive TME 4 plnts. The FEC-TME 4 (FEC-erive) plnts were use s CMD suseptile ontrol n wil-type plnts of TME 4 (WT-TME 4) n TME 7 (WT-TME 7) were use s CMD resistnt ontrols. The MTC-TME7 n MTC-TME 4 re meristem tip-erive plnts

10 Chuhn et l. BMC Plnt Biology (8) 8: Pge of n post-hrvest qulities [9] ut, s stte ove, this must our without losing the ritil trit for CMD resistne. Efforts to evelop plnt regenertion systems tht irumvent the nee for somti emryogenesis resulte in the ulogeni system reporte reently y Chuhn n Tylor []. In the present stuy, plnts of the CMD-type ultivrs TME 7 n TME 4 regenerte though this ytokinin-se shoot regenertion proess were hllenge with geminiviruses. In oth ultivrs, proportion of the regenerte plnt lines were onfirme to hve lost resistne to CMD. This response ws uniform n stle ross lonl replites of given line, suh tht ll plnts of regenerte resistnt line remine resistnt, n those tht were suseptile remine suseptile. Dt from plnts regenerte through ulogenesis n meristem tip ulture provie ler eviene tht somti emryogenesis per se is not the unerlying use for loss of resistne to geminiviruses. Unlike somti emryogenesis, shoot regenertion using met-topolin oes not involve exposure of tissues to high levels of uxin, nor the somlonl vrition ssoite with suh ulture systems. It remins unknown how loss of resistne ours, n why 7 6% of plnt lines regenerte vi ulogenesis lost resistne, while others remine fully resistnt. A possile explntion is tht isruption of shoot meristem integrity my e n unerlying ontriutor to loss of CMD resistne in CMD-type regenerte plnts. To test this hypothesis, meristem tip ulture ws investigte in CMD-type ultivrs. In this se, smll perentge (5 %) of plnt lines regenerte from oth CMD-type ultivrs (TME 7 n TME 4) were foun to hve lost resistne to CMD. These plnts were suseptile even to reltively less virulent strin of ACMV-CM, in the sme mnner esrie previously for somti emryo-erive plnts [4]. An lterntive hypothesis is tht epigeneti hnges our s result of morphogenesis in CMD-type ssv ultivrs. Suh hnges my ffet resistne gene(s) n/or suseptiility genes t the CMD lous. Inee, it hs previously een shown in five ssv ultivrs tht the meristem-erive plnts were epigenetilly ifferent thn the fiel grown plnts [5]. Aitionl stuies re unerwy to test these hypotheses. Conlusions The informtion presente here hs importnt implitions for iotehnologil pplitions in ssv, n efforts to eluite mehnisms of resistne to CMD. Non-CMD-type ultivrs re not ffete y pssge through somti emryogenesis or other morphogeni systems, n n therefore e use with onfiene s trgets for trnsgeni n gene eiting enhnement n mss propgtion through tissue ulture. Seonly, regenertion vi ulogenesis provies potentil solution for generting moifie CMD-type ultivrs tht retin resistne to CMD. However, plnts regenerte in this mnner require testing for their resistne to geminiviruses to eliminte those tht hve een ompromise. Finlly, meristem tip ulture shoul e use with ution when pplie to ssv ultivrs rrying the CMD-type mehnism euse it is possile to lose resistne to CMD in plnts reovere through this regenertion system. As for ulogenesis, regenerte plnt lines shoul e teste empirilly to onfirm tht CMD resistne is fully funtionl efore issemintion to frmers or estlishment in germplsm olletions. Loss of resistne through three ulture systems provies powerful toolset for investigting the moleulr mehnism ehin CMD resistne. The informtion esrie here will e ritil for esigning experiments n interpreting genomi, trnsriptomi n epigenomi tsets fouse on suh efforts. Aitionl file Aitionl file : Figure S. Response of orgnogenesis-erive plnts of ssv to inoultion with n infetious geminivirus lone of EACMV- K n MeSPY VIGS. EACMV-K (left) n MeSPY-VIGS (right) hllenge plnts of miropropgte TME 7. EACMV-K (left) n MeSPY-VIGS (right) hllenge FEC-erive plnts of TME 7. & EACMV-K (left) n MeSPY-VIGS (right) hllenge orgnogenesiserive plnts of TME 7. e EACMV-K (left) n MeSPY-VIGS (right) hllenge plnts of miropropgte TME 4. f EACMV-K (left) n MeSPY-VIGS (right) hllenge FEC-erive plnts of TME 4. g& h EACMV-K (left) n MeSPY-VIGS (right) hllenge orgnogenesiserive plnts of TME 4. (PPTX 7459 k) Aknowlegements We thnk the tehnil ssistne provie y Jenny Trn, Stephnie Lm, Dnielle Streth, Jennifer Winh, Clire Alin, Collin Leuert, Pul Butts, Jkson Gehn n Theoore Moll. Funing This work ws supporte y the Bill n Melin Gtes Fountion. The funing oy h no role in the esign of the stuy; olletion, nlysis, n interprettion of t; or in writing the mnusript. Avilility of t n mterils All t generte n nlyse uring this stuy re inlue in this pulishe rtile n its Aitionl file : Figure S. Authors ontriutions RC n NT oneive the experiments. RC, GB n NT esigne the experiments. RC n GB exeute the experiments. RC n NT wrote the mnusript. All uthors re n pprove the mnusript. Ethis pprovl n onsent to prtiipte Not pplile. Consent for pulition Not pplile. Competing interests The uthors elre tht they hve no ompeting interests.

11 Chuhn et l. BMC Plnt Biology (8) 8: Pge of Pulisher s Note Springer Nture remins neutrl with regr to jurisitionl lims in pulishe mps n institutionl ffilitions. Reeive: 5 Deemer 7 Aepte: 7 June 8 Referenes. IITA. Annul report (Interntionl Institute of Tropil Agriulture). In: IITA; 99.. Okogenin E, Moreno I, Tomkins J, Fuquet CM, Mkmilo G, Fregene M. Mrker-ssiste reeing for ssv mosi isese resistne. In: Vrshney R, Tueros R, eitors. Trnsltionl genomis for rop reeing: ioti stress, Volume. Chihester: John Wiley & Sons Lt;. p Okogenin E, Egesi CN, Olsnmi B, Ogunpo O, Khy S, Hurto P, et l. Moleulr mrker nlysis n vlition of resistne to ssv mosi isese in elite ssv genotypes in Nigeri. Crop Si. ;5: Beyene G, Chuhn RD, Wg H, Moll T, Alii T, Mino D, Crrington J, Tylor NJ. Loss of CMD-meite resistne to ssv mosi isese in plnts regenerte through somti emryogenesis. Mol Plnt Pthol. 6; 7: Kitimu SR, Tylor J, Mrh TJ, Tiro F, Wilkinson MJ, Roriguez Lopez CM. Meristem miropropgtion of ssv (Mnihot esulent) evokes genome-wie hnges in DNA methyltion. Front Plnt Si. 5;6: Ri IY, Hmlin MT, Kumr PL, Geil MA, Ikpn AS, Jnnink J-L, Kulkow PA. High-resolution mpping of resistne to ssv mosi geminiviruses in ssv using genotyping-y-sequening n its implitions for reeing. Virus Res. 4;86: Nunguru J, De Leon L, Doyle CD, Sseruwgi P, Plt G, Legg JP, et l. Two novel DNAs tht enhne symptoms n overome CMD resistne to ssv mosi isese. J Virol. 6;9: Oipio J, Alii T, Ingelreht I, Nusinow DA, Brt R, Tylor NJ. Effiient CRISPR/Cs9 genome eiting of phytoene esturse in ssv. Front Plnt Si. 7;8: Hummel A, Chuhn R, Cermk T, Mutk A, Vijyrghvn A, Boyher A, et l. Allele exhnge t the EPSPS lous onfers glyphoste tolerne in ssv. Plnt Biotehnol J. 7; [Epu he of print].. Chuhn RD, Beyene G, Klyev M, Fuquet CM, Tylor N. Improvements in groterium-meite trnsformtion of ssv (Mnihot esulent Crntz) for lrge-sle proution of trnsgeni plnts. Plnt Cell Tissue Orgn Cult. 5;: Chuhn RD, Tylor NJ. Met-topolin stimultes e novo shoot orgnogenesis n plnt regenertion in ssv. Plnt Cell Tissue Orgn Cult. 8;:9 4.. IITA. Cssv in vitro proessing n gene Bnking. In: IITA. ropgenenk.sgrp.gir.org/imges/file/lerning_spe/iit_cssv_ genenk_mnul.pf; 7.. Tylor N, Gitn-Solis E, Moll T, Trutermn B, Jones T, Prnjl A, et l. A high-throughput pltform for the proution n nlysis of trnsgeni ssv (Mnihot esulent) plnts. Trop Plnt Biol. ;5: Driver J, Kuniyuki A. In vitro propgtion of prox wlnut rootstok. Horti Si. 984;9: Murshige T, Skoog F. A revise meium for rpi growth n io ssys with too tissue ultures. Physiol Plnt. 96;5: Beyene G, Chuhn RD, Tylor NJ. A rpi virus-inue gene silening (VIGS) metho for ssessing resistne n suseptiility to ssv mosi isese. Virol J. 7;4: Bull SE, Brion RW, Sseruomwe WS, Ngugi K, Mrkhm PG, Stnley J. Geneti iversity n phylogeogrphy of ssv mosi viruses in Keny. J Gen Virol. 6;87: Ptil BL, Fuquet CM. Differentil intertion etween ssv mosi geminiviruses n geminivirus stellites. J Gen Virol. ;9: Fonong VN, Pit JS, Rey ME, e Kohko A, Behy RN, Fuquet CM. Eviene of synergism etween Afrin ssv mosi virus n new oule-reominnt geminivirus infeting ssv in Cmeroon. J Gen Virol. ;8: Fuquet C, Frgette D. Afrin ssv mosi virus: etiology, epiemiology n ontrol. Plnt Dis. 99;74:44.. Gresshoff PM, Doy CH. Derivtion of hploi ell line from Vitis vinifer n the importne of the stge of meioti evelopment of the nthers for hploi ulture of this n other gener. Zt Pflnzenphysiol. 974;7: 4.. Grout BWW. Meristem-tip ulture for propgtion n virus elimintion. In: Hll RD, eitor. Methos in moleulr iology, plnt ell ulture protools, vol.. Totow: Humn Press; 999. p Kwuki R, Priyo A, Amuge T, Nuwmny E, Ssemkul G, Tumwesigye S, et l. A reeing sheme for lol option of ssv (Mnihot esulent Crntz). J Plnt Bree Crop Si. ;:. 4. Ptil BL, Legg JP, Knju E, Fuquet CM. Cssv rown strek isese: thret to foo seurity in Afri. J Gen Virol. 5;96: Wg H, Beyene G, Aleu J, Oipio J, Oko-Okuj G, Chuhn RD, et l. Fiel level RNAi-meite resistne to ssv Brown strek isese ross multiple ropping yles n iverse est Afrin gro-eologil lotions. Front Plnt Si. 6;7:6. 6. Beyene G, Chuhn RD, Ilys M, Wg H, Fuquet CM, Mino D, Alii T, Tylor NJ. A virus-erive stke RNAi onstrut onfers roust resistne to ssv Brown strek isese. Front Plnt Si. 7;7:5. 7. Aenle A, Aworh O, Akromh R, Pryil G. Developing GM super ssv for improve helth n foo seurity: future hllenges in Afri. Agri Foo Seur. ;:. 8. Nrynn N, Beyene G, Chuhn RD, Gitn-Solis E, Grusk MA, Tylor N, Anerson P. Overexpression of Ariopsis VIT inreses umultion of iron in ssv roots n stems. Plnt Si. 5;4: Zinuin IM, Fthoni A, Surmonowti E, Beehing JR, Gruissem W, Vnershuren H. Cssv post-hrvest physiologil eteriortion: from triggers to symptoms. Posthrvest Biol Tehnol. 8;4:5.