III Vitro Germplasm Conservation of Atropa Baetica by Cold Storage
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l)o!a1l1c Gardens Micropropagalion News Volume 2 (3) Oclobcr 1998 III Vitro Germplasm Conservation of Atropa baetica by Cold Storage Manuel Cantos·, Rafael Zárale and Anlonio Troncoso Instituto de Recursos Naturales y Agrobiologia de Sev illa (IRNA S), CSIC, PO Box 1052,41080 Sevilla, Spain. Fax +34 5 462 40 02 • Author 10 whom correspondence should be addressed Abstract Gerlllplaslll 01" ;/1 v;tro derived plantlels of Atroptl baeticfl was lIlainlained by cold storage al 4°C in darkness for six 1Il0nths. Tite response of Ihe cold stored gcrlllplasm to growing culture co nditions was sludied on Murasltige & l l Skoog mediullI supplelllented wilh BAP (0.66 mg r ), NAA (0.24 mg r\ inosilol (100 mg r ), Ihiamine (1 mg 1'1) :Hul 3% sucrose durillg a six month periodo Tlle sur"iva! rate of Ihe germplasm was similar lo lile control (pl:lllt m:rterial growll under normal culture conditions), with all average pcrecnt ¡¡ge of 97.9 over the six month periodo The number of buds protlueed was similar for both control alld eold stOl"ed germplasm wHh an average 4.53 buds per explanl during Ihe six month period o Similarly, the number of shoots per ex plant did not differ betweell eOlilrol :lIId treatmenl. However, shoot length of eold stored gerlllplaslll \Vas slatistically shorler for Ihe 1" all(l 5'- monlh although Ihe ovcrall six month measurcmellts showcd 11 0 dirrcrcllec. Introduction Considerable interest has beeo showo in recent years Oll Ihe application of li ssue culture techoology for Ihe sloragc of plant gennplasm. Several strategies can be applied lo slow growlh to maintain planl gcrmplasrn, for examplc: Imlllipulating Ihe basal tll cdiulll, lowcring the optimal lIutrienl levels; ahering the physical conditions such as ternperature, ligh! regime alld gas allllosphere; application to Ihe medium of growth retardants (e.g. absicic acid) or osmoregulators (e.g. sorbitol, rn annitol) (Dodds & Ro berts, 1985). Altematively, cryopreservation has beell developed as a suilable rn ethod for long term gennplasm conse rv alion, Ihrough cessation of ce ll division , ¡hus avoiding Ihe possibilily of genetic vari ati on through il! ¡litro ce l1 di vis ion (Engel man, 1991). ·nlis techn ique howevcr necessitates specific Icchnica l equipment, nOI rcadily available in many laboratories. TIlC use of slow-growth conditions by lowering the temperalure is more comm onl y employed . Mos! cslablishcd slow-growth prolocols have bcen applicd to crop spcc ies (e.g. Doríon et al., 1994; Bencrjee & de Langhc, 1985) and few re ports exist 011 Ihe germplasm conserv ation or endangered plant species (e.g. Iriondo & Pérez, 199 1). Atropa baelica is a herbaceous perennial plant bclonging 10 tite Solanaceae famil y. The species is cndcmic to southern Spain and northem Morocco, its populalion is greatly reduced and it is now classified as an cndangered species (Hernándcz-Bermejo et al., 1994). Recently, a rapid in vüro propagation l1l ethod has heen reported (Zárate el al., 1997a) and ils Iropane alkaloid content detemlined (Zárale el al., I 997b). The use orslow-growt h itl I'ill·o slorage al 4"C in Ihe dark for Ihis species is Ihe subject oflhis sludy. Matcrials and Mcthods : Plall/ ma/eria! am! g rolV/h medium Forly eigbt explants of A. baelica lakcn frol1l established in vi/ro cultures (sce Zárate et al. 1997a) were elllploycd as a SO l1rce ofplanl matcrial. These consisted of stem scgmcnts of approximately 1 cm in lengtb bearing an axil1ary bud . The growth mediurn was composed of Murashigc and Skoog (1962) basal medil1m supplcmented with BAP (0.66 mg rl), NAA (0.24 rn g rl), inosilol (100 mg r\ thiaminc (1 mg rl) 3% sucrose alld solidified with 0.8% agar, ¡he pH adjustcd to 5.7 and al1t oc lavcd al 121°C for 15 mino Glass lest tubes wcre used as culture vcssels, cach containing lO mI ofmedium, covered wilh a plastic cap and sealed with Parafilm. 37 OOlanic Gardcns Micropropagation News Vol umc 2 (3) Octobcr 1998 Germplllsm slom ge llnd culture conditions Explanls wcre subcul tured on Ihe above mediulll and in iliall y placed in a culture su ile under nomlal growth cond itions 2 l (NGC) (i.e. 25 ± 2°C, 16 h. light, photoirradiancc 30l-lE m- s· ) for 15 da ys. 11lis was carried out lo reduce Ihc strcss li kely 10 occur if Ihese were direclly placed in Ihe dark cold room immediately after subculture. Ancr tbis period, 24 lubes conlaining Ihe cxp!anls wcre Iransferred lo a dark cold room (DCR) al 4nC and kept Ihefe for six montbs; the remaining 24 tubes growll under nonnal culture conditions. Eaeh monlh 4 tu bes from Ihe eold room and another four from Ihe culture sui(e were taken and explants excised and subcultured onto fresh Illedium ¡¡nd grown under NGC for 30 days. Afier Ihis period, the survival rate, bud and shoot induction, as well as shoot Icnglh wcre assessed. Slatisti ca l analyses of data were perfoffiled using Studcnt's I-test and Chi-Square lo assess difTcrenccs between Ireatments. Rcsults and Discussion Surviva! ra te of cxplanls fro m both control alld dark cold room conditions were statislica lly similar for each sampling lime (Fig. 1). llle mean values were 94.58% and 97.9 1% respectively, a high survival ra te which secms to indicate the sui tability of this plaul material for slow-growth storage conditions. "lllC nU lllber of buds induccd in explanls takclI from DCR or NGC was simil:lr for the 1", ti", 4'h and 6'h monlh (rig. 2), with a difference noted for the 3'<1 and 5'h months. Neverthcless, the mean number of buds produced afler thc six month period was si milar for Ihe control aud eold stored material, wüh valucs of 4.08 and 4. 53 respeclively. 111e number of buds produced by cold stored explanls inc reased sleadily up 10 Ihe 3rd monlh, showing a decrease afler this poinl. lllis is probably due lo the delerioralion of Ihe malerial and rcduced vigour aner a long lerm under slow-growth conditions" Explants from NGC show a steady rale ofbud formalion, although after six months this decrcased, probably due to nU lrienl depletion as Ihe medium agcd, afTecting the coudition of!he planl malerial used. lllese resulls show a clear regeneratíon polential for this species and suggests the efficiency of the slow-growlh slora ge condilions. This is in agreement wilh Wilkins et al. (1988), who also reported comparable results wilh fmi t tree species treated similarly. Fig. l.- Survival cate of Atropa baetica plantlets after six Fig. 2.- Numbe r of buds produced by Atropa baetica months wilhoul subcultu re. plantl ets from NGC and DCR over a six month periodo c.-.;,iooo -~ 8 ~:--'--...... , ......_ ... - '00 0.>''''- -~ 'E ro '-" w" ~ ", ..*, 00 '"m '¡; 'E b ' ;¡;T , D ~ " ,E " " o ~ Time (momhs) Time (monlhs) By co mparison, in assess ing Ihe number of shoots per explam (Fig. 3), there did not appear to be a rn arked difference over time for either of these Ircatmcnts. Furthermore, thcse values were gencra ll y similar, exccpl for the 3rd and 5'h months, indicating 11 0 deleterious cffcet ofthe DCR conditions upon the subsequent deyclopmcn! of Ihis species. "lllC cornposition of the growlh rn ed ium, particularly the presence of cytokinin, may have more effec t than enviro nmental conditions. Shoot length for cold trcated explants was sign ilicantly lower for the l ~ and 5'h month. The other fOUT samplings showcd no differCllce wilh an average shoot length of 2.7cm (Fig. 4). At Ihe 6rh month , shoot length va lues werc 10w for both control and Ireatmen!, which may suggesl a deleterious effecl of the six mon!h period on Ihe vigour oflhe plants. 38 Ootanic Garoens M icTOjlfOJl383 tion News Volunte: 2 (3) Octobc:r 1998 Fig. 3. - Number of shools per explant of Alro!,a baelica Fig. 4.- Shoot length of A/ropa baelica planllels from planl malerial laken from NGC and DCR over a six NGC and DCR over a six monlh periodo Eaeh val ll e is monlh period o Eaeh value is Ihe mean of al leas! 4 Ibe mean ofal lcasl4 replieates ± S.E. rcplieales ± S.E. j;r; , . Time: (monlhs) Time (ntonlhs) 11)e reslIlts presenled here show the praeticality of slow-growth stora ge of A. blletica al 4"C in the dark for up lo six monlhs withou l subeulturing. Nevertheless, after six monlhs sorne detrimental effeet upon Ihe growth and devcloprnenl of ¡he germplasm was observcd regarding number ofbuds and shool length (Figs. 2 alld 4). This effeel was also showll by expl:lnls cultured under NGC, and thcrcfore does not sccm 10 be rel:ued 10 Ihe DCR storage eonditions. A shorter storagc peTioe! would probably result in improved subseqllent gTow th oflhe explanls. Rcfcrcnccs Benerjee, N and de Langhe, E. (1985) A tissue culture ¡riondo, 1M. and Pérez, C. (1991) In v;tro slorage of technique fOT Tapid clonal propagation and slorage under three endangcrcd species from S.E. Spain. Bot. Gar. minima[ growth conditions of Musa (banana and Microp. News 1: 46-48. planta in). Plant Ce ll Rep. 4: 351-354. Murashigc, T. and Skoog F. (1962) A revised lIl ed ium Dodds, J. H. and Roberts. L.W. ( [985) Experiments in for rapid growth and bio-assays wilh lobacco tissue Plan! Tissuc Cultures. Cambridge Un iversi ty Press, culrures. Physiol. Planl., 15: 473-497. Cambridge, UK. Wilkins c.P. , Newbury H.J. and Dodds 1.1-1.