Arias Garzón, D.I.

Chávez, R.

Roca, W.

Williams, T

Withers, Lyndsey A.

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"j@JJiJ &l~~L ESTABLISHMENT AND OPERATION-OF A PILOT IN VITRQ ACTIVE GENEBANK OF CASSAVA COLEC~ON HISTORICA lb eL \ I I , I i I I ¡ I I \ I I , I i I 1. Bioteenology Researeh Unit, CIAT A.A. 6713 Cali, Colombia 2. IBPGR Rome, Italy 1. INTRODUCTION '------_1 Altt-.ough often vital adjunets as breeders' eolleetions, many existing tissue eult~lre eolleetions do not neeessarlly eonsti t~!te genebanks SLIÍ tabl e for long-term eonservatlon 01 germplasm. In this eontext, the lePGR has been laying the theoretieal fOLlndations for tMe development of iil vitro teehniques for the eonservation of eertain crop species, and it was necessary to test out the. theoretlcal framework developed. TMe IBPGR has assigned to CIAT worldwide responsibility for cassava (Manihot esculenta Crantz) germplasm eonservation. CIAT's stock, at the present, ineludes a field eolleetion of 4500 aecessions, a in vitro eolleetion of 3500 accessions, and approximately 1100 aeeessions maintained in the form of sexual seed. The principIe of genetic eonservation that material ,retrieved shoud represent the materi al aceessed has cC\Llsed eoncern abou~ the use of in-vitro systems because of the release of somaelonal variation. As a eonsequE'nce of genetie instability there can be variation ehromosome number and structure and in morphologieal and blochemlcal traits. It· has also been $uggested that eertain in vit".o ',,:·-,r"ge c:onditions can put germplasm integrity at risk beeaL'~" of directional genetie ehange in response to seleetion. This project pays speeial attention to this issue, by lc,~:·irlg at the extent to whieh this variatlon can oceur r-in; ;_; :,'_-'-:.9. storage of organi¡:ed strLlc:tures, and he", thlt "ri¿"" c. .-, be detected by u.ing morphologieal, cytol09ic&: _~G biochemical criteria. 2. OBJECTIVES The overalI objeetive of this IBPGR-CIAT CDlli~C·2tivF projeet is to assess and demonstrate the teehn:el.l ~-.j logistical aspeets of establishing and operating an in ~L:~~ Active Genebank IP.IVAGI aeeording to standards set by IB?GR and CIAT uBing eassava (Manihot escL!lent¿) as " model crop. 3. GENERAL WOR¡< PLAN Using the e"perlenee gained at CIAT 01"1 in ,'itro eonservati 01"1 of eassava elones and the reeoffiffili!ndClt i on of. IBPGR 01"1 monítoring genetic stabníty of root. erops, the majar steps reqLtired for the establishing and runnÍng of a pilot-IVAG wara outlined for a period of three years. The major steps are: a) seleetíon, sampling and .initlal ehélraeterizéltion of clones, b) introduetion of clones lnto in vi troeul ture, sLlbeLll ti .• r i ng élnd i ntensl ve mieropropagation, e) in vitro storage under minima! conditions of growth; élnd d) monitoring of genetie stability. At present the first two steps mentioned aboye have already been performed. 4. SELECTI ON OF GENOTVPES FOR l.I:i§;. E=) VAG. The eassava germplélsm being used in the P-IVAG eomprises 100 clones selected fram a total of 4::,00 aeeessions eurrently maintained at CIAT 11"1 the fleld genebank. Selection was made mélinly 01"1 the basis of phenotypic variation, so interclonal differences and morphotypes cOL.I d be easi 1 y recogni z ed redL.ci ng the number of morphologieal duplieates. Hopefully the clones ¡¡¡Mould represent a wide range of the genetie variation present in the cassava gene pool. In addition to morphologíeal variation, three other criteria were taken int~ aceount for elonal seleetion: a) adaptation to the main edaphoelimatie (eeosystemsl zones of eultivated eassava, bl geographieal latitude, longitude, altitude and eountry of or1g1n; and el a few elite clones with outstanding yield potential. 5. SAMPLING OF MATERIAL Five plants per seleeted clone were labeled in th.: field g~nebank. Four stem euttings Istakesl were takEn from each one of the labeled plants, í.e. twenty sta~e~ pEr clone, from whieh five stélkes were planted in the Assaei2~ed Field Genabank IAFG) and fifteen were planted !n ~he glasshouse for disease Inde"ing, Electrochc"0 le charaeterízation, tharmcterapy and meristem tip cwl~,·e. Due to the large work load and spacp requir~~ du~~rG t~e initial sampling and processing st~ges the nI2~F'-:¿J ~~s handled as five lots e;;¡eh compriEí ng twe,-,ty ele,""'-, ,' .•. , " 4-5 weeks interval lots. The lineage loentlty o{ c.~'. plants and stalces are beíng kept dC\ring the O\Jl 5~·"~". c"' sampl ing and introduction o;: m¿,;teritRl to i..D. V) ::::.s: Cl., ~ ,,:::..;.-¿:. and storage. 6. DISEASE INDEXING The phytosanitary conditions of mother pla~ts W2S determined prior to thermotherapy and merlstem-tlp culture using a combination of symptomatology, graft lnocul2tlon, ELISA and dsRNA analysís for the deteetion of eassava common mosafc vlru~ ICCMVI, cassava X virus ICsXV), the . frogskin disease (FSD) , caribbean mosalc disease (CMD) and cassava latent-di~ease (CVL). FSD root symptoms were observed in 13 of the 100 clones. Eleven clones werepositive by graft inoculation to the susc:eptible clone "Secundina". CCMV was not detected in any of the clones bythe ELISA test. However the ELISA test -did detect in CsXV in five clones. Virus-specific dsRNA was detected in 52 clones of the 100 clones tested. AII clones testing positive fer virus will be retested after thermother.r;py and meristem-tip c:~,lture, and prior to in vitro storage. Shol.,ld any of the clones remain positive following treatment, mic:rothermotherapy wil1 be carried out in vitro. 7. IN VITRO CULTUF:E AND STORASE Thermotherapy of the 500 mother pl<.>"ts was carried OL,t in the growth C:hamber, exposing plants to high temperatures 35-40 oC at an illumination of 3.000-5.000 lu:-, , for 2-3 weeks. Meristem tips were e>:c:ised from fast growing shoots and placed in culture medium developed at ClAT. Subculturing of plantlets and subsequent micropropagation were made in medium 17 N until eno~'gh replic:"tes were obtained for evaluation and for storage under mini,,,imal conditions of growth, u$ing the medium 8S. Up to the present all clones from the first group have been introduced into in vitro storage. A tot"l of 25 individ~,als wil1 be maintained in test tubes for the P-IVAG (fig.1) Thre. markedly different clones, from the genetic view point, each with 25 replicates, are being used as c:ontrols for monitoring the effect of subculturing frequency during storage and the effect of meristem origin, either apical or a>, i 1 ar. 8. MORPHOLOGICAL CHARACTERIZATION Preliminary mo..-phologic:al evaluation was: mare in the originai fi.ld genebank from whic:h the m.teri~l for ~he P-IVA6 was selected. More detailej marphologiC&l ev¿lustion was carried out in the A!>sociated Field 6er,e:O '" ,<l. (AFe;' ","ere growing conditions are more u"lfcrffi~ R~CL'Jt~ o~ t~lS evaluation indicated a wlde ranQ8 c+ genetic v~r·.~~il~t~' ·~r most of the internationally rE¡:c9~ized C~.S2~a d~~~j-:~t=rs among these lOO clones, represaontlf,g thE' spectru,." ¡;¡" 9""c-::c: vari ati on wi thi '1 the c:rop speci es, t"g~:_Lh01 §:Zc<;:~_:_. ino s eore c~ssava germplasm a]so dlEpl¿ys An e~¿10:Lic~¿-y pathway of the crop ranging from ver y primitiva culti~a·E to advanced elite c:lones. A total of 62 descriptors were recordad genotype, out of which 21 are passport d~ta, for ",nc E2 .. :h the remaining compriSfrs discreta morphologlcal tr-aits, inde::ln::¡, of flowe'-lng f.eld charts qualltative and quant.tatlve, disease electrophoretic patterns and pollen fert.lity clones. The information was recorded in especially designad for use 1n field and for direc:t transcription to the computer. Charac:ti01rizatian of growing plants both in the field genebank and in the AFG has beem done on six months old plants. Leaf shape, size and color was mainly studied on the 11th leaf starting from the topo 9. ELECTROPHORESIS Isozyme electrophoretic methods have been applied lo the characterization and dlscrimination of the 1('0 selec:te-d genotypes. Isozyme clustering data 0+ 16 ident: ~ied major bands shows an association of the 100 clones in 32 cluE~er.d groups and enabled the identification of six duplicates among the material. Electrophoregrams fram the 100 clones were computer analyzed; up to the prese¡t 10 alfa 2nd 6 beta esterase bands were :den~iflEd to diff.~entiate eAch genotype. Seventeen elonE's sho.,ed c:or,;:il s:ler,t 2.nd '.c,1. 1 defined el ectrophoregrams , from which, the pattern of ev. MBRA309 was taken as a reference t.o compare with all banc,ng patterns from the core germplasm. Out cf thlS group 3 clones wer-e selected for use in a related e>:periment to test the effect of subculture frequency on genet1c stability. Clustering data of alfa and beta esterases are belng supplemented with the data obtained from the new LKB ultrascan XL laser densitometer that overcomes the difficulties of accurately quantifying electrophoretlc gels. Beta esterease band, most comon to the eDre cassava germplasm, was used as a reference diring the scanln of gels. The comparison of ultrascan XL and clustering o' electrophoregrams will give us incomparable help for further moní tor i ng of the genet i c stab i 1 i t Y on iD. :::::.iir:.Q c .. 'l tures. 10. ASSDCIATED FIELD GENEBANf: (AFGI The AFG represents, sI the original core C~Esava germplasm without treatment under any in vitrCJ_ cc-¡dic.l0n, and bl the field plot with plants origlnate fro~ ln-vl~ro conditions. They are used syst.m?~ic.lly fo- c:onsl evaluation and as .'contrel f~~ eveluati~g t~c ~-I~J~'~ ~o compare any possible ptlenotypl<:: chO,;-,g'2 i" ~_",:-S;" .2",":1 clones undE'1'"" minim2.1 C:O~¡C::~_lO:-S o.;. ~""'ovJ";::"¡. (I:~~-ll; -j di serete morphologic~l tri,:ut~. St2J:E? S¿;{,"¡¡:JE-:; _ ..... :,.::. '-¿--'1 identified plant growirl9 lr¡ the 'Lield S~;"'H:C.'¿~··~ h'~-"'~ :'i-' I~! and planted in 28 x 60 ro 2 plo~. Five ~1¿nt5 ~~'- =~~~~ Wf?~ planted in a dista:nce t,etwcE'n ;;lants 1 m, bS''!:.~ .. eiE':-: Y";:',4<:-.:. rn and between 1 ines 3 m~ Fl¿!""lts are g-c\~r .. r¡~ ll~-,;:;;:'" . ~ conditions and are ~lso di<:¡::-li'ying contré<Sélr.g mQ,,-~,-,::jc;"c,,"] features. 11. DATA BASE A c:omputeri::!ed data base on chare<cte:rl::~tlcn L·-f morphological traits, disease indexing and blochEmlcal dl-a has alreadl begun. A software package ,. being dev.lop~d , far running all _Internatianal Data will be used with for -the F'-1VAG. aspects of the P-IVAG Llslng the Management System I1DMS). This software the ne~ hardware to be purchased very soon 12. FACILITIES FOR F:UNNING THE P-IVAG Thefollowing CIAT laboratories and other fac:ili hes hav!? been used for establishlng and operating the P-IVAG. 1-2-3-4-5-6-7-8-9-10-11-12-1-,. ..,. Tissue culture lab. Trar,sfe..-ence room .i.tn vi troj Propagation room (In v¡trol le!. Vl trr:~ storage room Cytology lab. Bioc:hemistry IElectrophoresis) lab. Vi rol ogy J ab. CompLlter Center Photographic: and library facilities Glasshouses Sc:reen HOl.lses Field plots STAFFING Becal.lse the comple>: nature of the P-IVAG project a lot of intel'"action is involved and remarkable help, callabo ... ation and ódvice ha!;:, been received from severa! CIAT scientists, assistants, technicians and adminlstrative personnel. It i s mer,ti oned onl y the m",i r, staff 0+ thl? P-IVAG: a. Principal Rese?rch Staff .o.... R. Chavez D.... W. Roc:a b. Reseal'"c:h As~js~2nts Miss D. 1. Ana,; (fulltl1r.c» Miss CataRo (p .... tt,me) c. Tec:hni c:i?r M .... QUH "ro (+L'll ti ".,,,) MI'" • R. A..- i ¿,!'; ( .; c·' 1 tI r' - ) d.. Ccll at.QUt o!:,_E __ C'::CI.f: __ .::..:td5 !~;r2 Dr. C. Her"!i.hE- y (C?ss2,",/a Frogr¿·.m) Dr. O. Wood (Genetic Resowr'c:es Uritl D..-. M. Halle (IBFGR) Dr. B. Nolt (Cassava P..-ogram) Dr. L. Ch~pas (Data Servlces Unit) Dr. L. Withers IIBPGR) For more detailed information Q!1 F-IV?',G ?c:tlVlty, ccr,téC-::: R. Chavez /W. Roca. Centre lnt.~n~cicnal de Agri~LlltL'ra Tropical (CIAr) A.A. 6713 C21i-Colombi •• ", I ' , I Fig. 1. Storage of cassava clonas urdpr min!m&l ccnditio~. of grcwth (P .. IVA5). Li~ ¿n2- ML\rOZ, LE~'. lé'::r,;-¡~c:1,~~ from the BiQte=h~cJcgy RRs •• rch U~:t-CJ~T helee • ~~.­tube NO' .. 2~ [ar .t21nlng a C¿,'J>:JvéJ :in=~~itt~_~;:. c.ul'[.~"t::. • I I II THEORET!CAL ANO APPLIED GENETICS (TAG) 1988 (J n press) 2 i 3 Transfer of Resistance ta PLaV Titer Buildup fram Solanum ecuberosum to a • Tuber·bearing Solanum Gene Pool R. Chavez1 • c. R. anc M. • • 1 Biotechnology Research Unit. Centro Interr.acior.al de Agricultura Tropical lQ (CIAT) A.A. 6713, Cali, Colombia 11 2 USDA.Agricultural Research Service, Irrigated Agriculture Research and 1 Extension Center, P.O. Box 30, Prosser, YA, 99350, USA 1 3 International Po tato Center, Department of Germplasm Resources, 1 APDO 5969, Lima, Peru 1 1 1 1 1 2 2 2 2 2. • • DA MANUSCR'" CUtDE SHEET 

Establishment and operation of a pilot in vitro active genebank of cassava

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Establishment and operation of a pilot in vitro active genebank of cassava

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