Plant Genetic Resources newsletter No. 124, December 2000

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Plant Genetic Resources Newsletter

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Cover: Cocoa fruits. This crop is dis-cussed in the paper by Lachenaud (pp.1-6). Photo by Jan Engels, IPGRI.

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Portada: Frutos de cacao. Se hablade este cultivo en el documento escri-to por Lachenaud (pp. 1-6). Foto deJan Engels , IPGRI.

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Plant Genetic Resources Newsletter, 2000, No. 124 1Plant Genetic Resources Newsletter, 2000, No. 124: 1-6

Agronomic assessment of wild cocoa trees(Theobroma cacao L.) from the Camopi andTanpok basins (French Guiana)Ph. Lachenaud1*, G. Oliver2 and Ph. Letourmy3

1 Cirad-Cp, 01 BP 6483, Abidjan 01, Ivory Coast. Email [email protected] Cirad-Cp, BP 701, 97387 Kourou Cedex (Guyane), France3 Cirad-Ca/Mabis, TA 70/01, Montpellier Cedex 5, France

SummaryAgronomic assessment of wildcocoa trees (Theobroma cacaoL.) from the Camopi andTanpok basins (French Guiana)A study was made of around 1600 indi-vidual cocoa trees (Theobroma cacao L.)representing 144 open-pollinated prog-enies and 11 wild populations identifiedin two river basins of French Guiana,based on the following selection criteria:juvenile growth (or vigour), adultvigour, the yield:vigour ratio, earlinessof production, yield, average pod weightand losses due to rot in the field. Obser-vations were carried out at CIRAD’sSinnamary station in French Guiana overa total of 10 years. The analysis of vari-ance carried out on the seven most nu-merous populations (amounting to 97%of the individuals) revealed populationand/or progeny effects for all the crite-ria. It is proposed that the study materialbe incorporated into genetic improve-ment programmes, particularly for yield,the yield:vigour ratio and performancein the field with respect to rot diseases.To that end, practical indications aregiven to breeders for choosing from thepopulations and progenies.

Key words: Characterization, descrip-tors, French Guiana, genetic improve-ment, Phytophthora, populations,Theobroma cacao, wild cocoa trees

Abbreviation: CIRAD: Centre deCoopération Internationale enRecherche Agronomique pour leDéveloppement (Montpellier, France)

ResumenEvaluación económica decacao silvestre (Theobromacacao L.) de las cuencas deCamopi y Tanpok (GuayanaFrancesa)Se hizo un estudio de unos 1600 ejem-plares de árboles de cacao (Theobromacacao L.) representativos de 144 linajes depolinización abierta y 11 poblaciones sil-vestres identificados en dos cuencas flu-viales de Guayana Francesa, con los sigu-ientes criterios de selección: crecimiento(o vigor) juvenil, vigor adulto; relaciónrendimiento-vigor, precocidad de pro-ducción, rendimiento, peso medio de lavaina y pérdidas por podredumbre en elcampo. Las observaciones se realizaronen la estación Sinamary del CIRAD en laGuayana Francesa a lo largo de 10 años.El análisis de varianza realizado en lassiete poblaciones más numerosas (quecontienen el 97% de los árboles) revelólos efectos sobre la población y/o el lina-je según todos los criterios. Se proponeque el material del estudio se incorpore aprogramas de mejoramiento genético,en particular en atención al rendimiento,la relación rendimiento-vigor y los re-sultados en el campo respecto a las enfer-medades de podredumbre. Con ese fin,se dan indicaciones prácticas a los fitoge-netistas para escoger entre poblaciones ylinajes.

ARTICLE

RésuméEvaluation agronomique descacaoyers spontanés(Theobroma cacao L.) desbassins du Camopi et duTanpok (Guyane Française)Environ 1600 cacaoyers (Theobroma cacaoL.) représentant 144 descendances libresappartenant à 11 populations naturellesidentifiées dans deux bassins fluviaux deGuyane Française, ont été étudiés indivi-duellement pour les critères de sélectionsuivants : la croissance (ou vigueur) juvé-nile, la vigueur adulte, le rapport produc-tion-vigueur, la précocité de production,la production, le poids moyen d’une ca-bosse et les pertes par pourritures auchamp. Les observations ont été réal-isées à la station CIRAD de Sinnamary enGuyane, sur une durée totale de 10 ans.L’analyse de la variance, effectuée sur les7 populations les plus nombreuses(représentant 97% des individus) permetde mettre en évidence des effets “popu-lation” et/ou “descendance” pour tousles critères. Il est proposé d’intégrer lematériel étudié dans les programmesd’amélioration génétique, particulière-ment pour les critères de production, derapport Production / Vigueur et de com-portement vis-à-vis des pourritures auchamp. Dans ce but, des indications pra-tiques sont données aux sélectionneursquant aux populations et descendances àpréférer.

IntroductionThe wild cocoa trees of southeastern French Guiana, whichhave been known to exist since 1729 (Capperon, 1731; Leconteand Challot 1897), have been surveyed and collected on threeoccasions in 1987, 1990 and 1995 (Lachenaud and Sallée 1993;Lachenaud et al. 1997). Numerous studies of this germplasmsince 1987 (Lanaud 1987; Lachenaud and Sallée 1993; Laurentet al. 1994; N’Goran et al. 1994; Lachenaud et al. 1997; Sounigo etal. 1996, 1999; Lachenaud et al. 1999) have all revealed its unique-ness among ‘Forastero’ cocoa trees (T. cacao subsp. sphaerocarpum)(Cuatrecasas 1964). This uniqueness is such that this group ofcocoa trees is now considered one of the four poles of thespecies’ genetic structuring (Lachenaud 1997; Lanaud et al.

1999). Nevertheless, before it can be used for genetic improve-ment, it needs to be described, characterized and evaluated,particularly for selection criteria.

Agronomic and morphological characterization of this wildGuianan material has been under way since 1988 nearSinnamary, French Guiana, where the Paracou-Combi referencecollection is planted. The aim is twofold: to gain further knowl-edge of variability in this original material and identify origins(populations or families) that are potentially useful in breeding,and to provide practical indications for breeders who wish to usethem. This article covers only the latter aspect. It presents theresults of 10 years’ agronomic assessment of progenies from

2 Plant Genetic Resources Newsletter, 2000, No. 124

wild cocoa trees collected in 1987 in the basins of the Tanpokriver (upper basin of the Maroni river) and the Camopi river(upper basin of the Oyapok). The traits used, which are allselection criteria, were vigour, earliness of production, yield, theyield:vigour ratio, pod size and resistance to rot (caused byvarious species of Phytophthora) in the field.

Material and methodsPlanting materialThe planting material studied at the outset comprised 144 open-pollinated progenies of wild cocoa trees. Each progeny camefrom a pod taken from a wild mother-tree, and each mother-treewas represented by only one progeny. The 144 mother-treesoriginated from two subbasins in the far southeast of the coun-try (between 53°27' and 53°10' W and 2°19' and 2°23' N). Treescollected in the Camopi basin belonged to wild populations 1, 3,6, 7, 8, 9, 10, 11, 12 and 13, while those collected in the Tanpokbasin belonged to wild population 5 (Lachenaud and Sallée1993). The population concept used here was that used byPernes (1984) and Berthaud (1986) for coffee trees, and corre-sponds to the subpopulation (‘deme’) referred to by Hartl andClark (1997).

The number of trees per population (on planting and at theend of the study) is shown in Table 1. On planting, the numbervaried from 2 to 19 trees per progeny, with an average of 11.2.The trees were planted between January and June 1988 inseven blocks in two plots either side of a small bottomland,over a total area of 0.99 ha. The trees were planted 3 m x 2 mapart, corrresponding to a density of 1667 plants/ha. Thecocoa seedlings were planted under temporary banana shade(at the same density) for 4 years. The permanent shading wasprovided by Gliricidia sp., spaced 6 m x 6 m apart. The blocksreceived mineral fertilization of the ‘soil diagnosis’ type (Jadinand Snoeck 1985), supplemented with applications of nitro-gen and boron. Each block contained 2 to 11 populations andeach population analyzed was present in three to five blocks.The seven blocks were monitored for 10 years. The trial did nothave integrated controls representing other groups of cocoatrees, such as Upper or Lower Amazon Forasteros orTrinitrarios. Nevertheless, in some cases, orders of magnitude

could be obtained from the results of an adjacent hybridcomparative trial (plot C0) also monitored for 10 years, six ofwhich were contemporary with the study in question(Lachenaud et al. 1994). The edapho-climatic andphytosanitary conditions at the Paracou-Combi station havebeen described in earlier work (Lachenaud et al. 1994).

Agronomic descriptorsThe agronomic descriptors chosen for each of the trees were asfollows:l Juvenile growth, i.e. the increase in ‘collar’ cross-section (15cm from the ground) between 1 and 2 years in the field. This wascalculated (in cm2) from two diameters measured with slidecalipers.l Adult vigour. The circumference of the tree at 10 years oldwas measured 50 cm from the ground with a tape measure anda cross-section calculated (in cm2).l Yield. This was calculated from the number of healthypods, their weight and the number of rotten pods (withoutspecifying the causal agent) for each tree and each harvestinground. Earliness of production and overall yield were obtainedby cumulating the figures. Final yield was expressed as theweight of healthy pods, or as a potential yield if rotten podswere taken into account. The dry cocoa equivalent was calcu-lated by multiplying pod weight (healthy or total) by acoefficent of 0.0875, equal to 0.25 (ratio of fresh bean weight topod weight) x 0.35 (ratio of dry cocoa to fresh beans)(Lachenaud et al. 1994).l Yield:vigour ratio. This is the ratio of potential yield cumu-lated at 10 years to the cross-section 50 cm from the ground at10 years. It was therefore expressed as kg of pods per cm2.l Average pod weight. The average weight of one pod pertree was calculated from the ratio of cumulated healthy podweight to the number of healthy pods, keeping only those treesthat produced at least 20 healthy pods, a quantity whichenabled characterization of a tree for this criterion (N’Goran1994).l Losses due to rot diseases. The rate of rotten pods per treewas determined by counting. The analysis results were means(per population or per progeny) of individual rates, taking a

minimum yield of 50 pods per tree.This is quite a high value but wasnecessary given the low rotten podpercentages. A study of correlations(not shown) revealed that this pa-rameter was well correlated to theoverall rate per population or perprogeny (total rotten pods/totalpods).

Statistical methodsThe design of the collection lent itselfto an analysis in unbalanced incom-plete blocks. The analysis of variancewas carried out on data from micro-plots of varying sizes (from 1 to 10trees), which were progeny-block

Table 1. Distribution by population of open-pollinated progenies (OP) andtrees studied, on planting (1988) and after monitoring for 10 years (1997)

1988 1997

Basin Subbasin Population OP Trees OP Trees

Oyapok Camopi 1 26 274 26 2563 15 206 15 1876 1 5 1 57 20 209 19 1688 2 16 2 119 50 555 50 510

10 1 12 1 1211 2 21 1 912 10 113 10 10113 15 176 15 162

Maroni Tanpok 5 2 34 2 33Totals 144 1621 142 1454

Plant Genetic Resources Newsletter, 2000, No. 124 3

combinations. The fixed effects model used was as follows:Yijk = µ + Bi + Pj + Dk (Pj) + εijkwhere Yijk = the mean for the micro-plot of progeny k, population

j, block iµ = overall meanBi = effect of block iPj = effect of population jDk (Pj) = effect of progeny k in population jεijk = residual random error

The overall means per population, or per progeny, resulted froman adjustment to the blocks, and weighting by the numbers oftrees in the microplots.

The analyses of variance were carried out with the SASsoftware GLM procedure, using the LSMEANS option (SAS1989). In the absence of a way to ensure the validity of theanalyses of variance carried out in this particular case, themain criterion adopted was homogeneity of thewithin-population variances of the residuals (the raw residu-als multiplied by the square root of the numbers of trees in themicroplots), using Levene’s test at 5%. In the event of hetero-geneity, the responsible populations were excluded from theanalyses (Table 2). Normality of the model residuals was alsochecked using Shapiro-Wilk’s W test. However, owing to thelarge numbers studied and the robustness of the analysis ofvariance compared to the deviations from the norm, the formalnon-normality of the model residuals was not a problem,provided the distribution approximately followed a ‘bell’ curve.The results shown (cf . Table 3) were obtained by analyses thatverified those criteria.

If the population and/or progeny effects were significant,the adjusted means were compared (2 by 2 by a Student’s t-test)and a synthesis is shown (Table 2). For the progenies, given thelarge numbers involved, comparisons were made only with themean progeny (the progeny whose performance was equivalentto the mean of those progenies included in the analysis; theprogeny used varied depending on the descriptor), and themeans could be classed in three groups: worse than, equal to, orbetter than the mean progeny. Only the seven most representedpopulations (1, 3, 5, 7, 9, 12 and 13, i.e. 97.4% of the trees at 10years) were analyzed. In principle, progenies with fewer thanfive trees were always taken out of the analyses.

ResultsJuvenile growthThe analysis of variance revealed very highly significant block,population and progeny effects (probability > F = 0.0001). Theoverall model effect was also very highly significant (R2=0.89).Classification of the populations according to their adjustedmeans (in cm2) is shown in Table 2.

Twelve progenies were statistically inferior to the mean ofthe population, and 11 better. In increasing order of growth, thebest five progenies were GU 186, 238, 340, 287 and 264. Theworst five progenies, in decreasing order of growth, were GU291, 313, 350, 354 and 126.

Adult vigourThe analysis of variance revealed a very highly significant modeleffect (R2=0.81), along with block, population and progeny effectsthat were also very highly significant. The population means (incm2) could be classed in two groups (Table 2). Eleven progenieswere worse than the mean progeny, and 9 better. The five mostvigorous families were GU 163, 174, 285, 323 and 178, whilst thefive least vigorous families were GU 350, 304, 344, 345 and 313.

YieldGiven the strong correlations between the different yield de-scriptors (not shown), only the ‘potential’ variable was ana-lyzed. The analysis of variance revealed a very highly significantmodel effect (R2=0.75). The population effect was not signifi-cant, but progeny and block effects were very highly significant.Exclusion of population 7 from the analysis could explain thelack of a population effect despite the high amplitude of theadjusted means between well represented populations (from11.1 kg of pods for population 3 to 20.4 for population 7). Fourprogenies gave significantly lower yields than the mean prog-eny, and 14 gave higher yields. In increasing order, the fivehighest yielding progenies were GU 134, 280, 143, 303 and 285.The five lowest yielding progenies were GU 244, 250, 222, 113and 282.

Yield:vigour ratioThe analysis of variance revealed a very highly significant modeleffect (R2=0.73). The population effect was not significant (prob-

Table 2. Means (adjusted to the blocks) of seven main populations (and plot means) for seven traits. Thevalues indicated for potential yield, potential yield/cross-section and earliness are in kg of pods

Population Juvenile Adult Potential Potential yield/ Earliness Average % rotgrowth vigour yield cross-section (kg/tree) pod weight(cm 2 ) (cm 2) (kg/tree) (kg /cm 2) (g)

1 13.8 a 87.7 a 18.2 a 0.17 a 1.2 ab 435 a 1.3 bc3 13.5 ab 87.4 a 11.1 a 0.11 a 0.8 ab 322 d 1.4 bc5 13.4 abc 85.2 ab 14.9 a 0.14 a 2.6 * 351 c 3.6 a7 10.2 d 75.7 b 20.4 * 0.21 * 3.0 * 380 b 1.6 b9 11.6 cd 83.2 ab 15.7 a 0.15 a 1.2 ab 359 c 1.0 bc12 12.1 bc 88.1 a 16.1 a 0.17 a 0.6 b 316 d 1.2 bc13 12.7 abc 82.6 ab 16.9 a 0.17 a 1.3 a 386 b 0.6 cMean 12.3 85.1 16.4 0.16 1.2 367 1.2

* = population taken out of the analysis to satisfy homogeneity of within-population variances.Means followed by the same letter are not significantly different (P<0.05).


ably due to the exclusion of population 7), but progeny andblock effects were very highly significant. Six progenies weresignificantly worse than the mean progeny and 12 were better.The best five were GU 325, 139, 285, 303 and 134; the worst fivewere GU 313, 194, 250, 113 and 282.

Earliness of productionIn order to carry out a valid analysis of variance, the two mostprecocious populations, 5 and 7, had to be excluded. On theremaining sample (5 populations, 107 progenies), the analysisrevealed a very highly significant model effect (R2 = 0.70), asignificant population effect and very highly significant blockand progeny effects. Based on their (adjusted) yield the popula-tions could be classed in two homogeneous groups (Table 2).The best five were GU 146, 116, 139, 143 and 134. Twenty-eightprogenies (out of 143), 21 of which were represented by at leastfive trees, did not produce a single pod for five years.

Average pod weightConsidering only those trees that produced at least 20 healthypods, forty-eight progenies were excluded from the analysis ofvariance. The model was very highly significant (R2=0.88), aswere the effects considered, particularly the population effect(F=44.0). Classification of the means revealed four separategroups (Table 2). Sixteen progenies were significantly betterthan the mean progeny and 19 worse. The progenies with theheaviest pods were GU 157, 161, 274, 212 and 285. Those withthe smallest pods were GU 311, 162, 218, 205 and 230.

Losses due to rot diseasesFixing yield at 50 pods per tree substantially reduced the num-ber of trees to be studied, along with the number of satisfactorilyrepresented progenies (only 42 out of the 142 progenies had fivetrees or more). We therefore simplified the model, keeping onlythe population and block effects for the analysis of variance(and using arc sine square root transformation of the rotten podrate to normalize the residuals). The model was very highlysignificant (but R2 was only 0.18), as were the population andblock effects. The population means could be classed in threegroups (Table 2). The five progenies (with at least five trees) with

fewest losses due to rot diseases were GU 252, 321, 325, 134 and240 (from 0.52 to 0.13%). The progenies with the greatest losseswere GU 186, 157, 129, 146 and 116 (from 2.10 to 3.05%).

DiscussionThe analysis of variance revealed significant population and/orprogeny effects that, along with the degree of variability notedfor most of the traits, provide for effective selection. Althoughour results were obtained in only a single environment, theyshould help geneticists who have Guianan material at theirdisposal, or who wish to acquire it, to make selections. Tofacilitate that selection, Table 3 provides a list of the populationsand progenies, and Table 4 indicates the correspondence be-tween the material mentioned in this article and the sib material(half or full sibs, derived from pods collected from the samemother-trees) already supplied to several producing countries.CIRAD has been supplying GU clonal material on request tocountries or institutions from its quarantine centre in Montpelliersince 1989 (Lachenaud and Sallée 1993). More recently, thismaterial has been supplied from quarantine centres at the Uni-versity of Reading, UK and the Trinidad Cocoa Research Unit inBarbados.The following comments can be made regarding the main de-scriptors:l Yield:vigour ratio. Population 7 is the best population forthis paramount selection criterion in cocoa improvement (Lotodéand Lachenaud 1988; Paulin et al. 1993). Two progenies (GU 134and GU 303) gave better values than those scored by the best

Table 4. Correspondence between the GU progenies mentioned in this article (A) and those supplied toproducing countries and quarantine centres (B)

A B A B A B A B

GU 113 GU 114 GU 174 GU 175 GU 250 GU 251 GU 313 GU 314GU 116 - GU 178 GU 179 GU 252 GU 253 GU 321 GU 322GU 126 - GU 186 - GU 264 GU 265 GU 323 GU 324GU 129 - GU 194 GU 195 GU 274 GU 275 GU 325 -GU 134 - GU 205 - GU 280 GU 281 GU 340 GU 341GU 139 GU 140 GU 212 GU 213 GU 282 - GU 344 -GU 143 GU 144 GU 218 GU 219 GU 285 GU 286 GU 345 GU 346GU 146 GU 147 GU 222 - GU 287 GU 288 GU 350 GU 351GU 157 GU 158 GU 230 GU 231 GU 291 - GU 354 GU 355GU 161 - GU 238 GU 239 GU 303 -GU 162 - GU 240 GU 241 GU 304 GU 305GU 163 GU 164 GU 244 - GU 311 GU 312

- = no corresponding material outside French Guiana.

Table 3. Distribution of GU progenies by population

Population GU progeny

1 116, 156 to 161 and 250 to 2863 218 to 241 and 347 to 3495 113 to 116 and 1237 126 to 1529 162 to 198, 242 to 249, 297 to 330 and

350 to 35512 203 to 21713 287 to 295 and 331 to 346


progeny (UPA 402 x UF 676) in the neighbouring hybrid trial.Given its poor yield:vigour ratio, population 3 should be ruledout for further selection.l Earliness of production. The average precocity of the studymaterial was low and well below that of the hybrid material inthe adjacent trial. However, some blocks suffered considerablyfrom thrips (Selenothrips rubrocinctus) attacks in the first threeyears, which affected juvenile growth and probably delayed thestart of bearing. This may explain the low correlations seenbetween the juvenile and adult criteria. The most precociouspopulation was 7, followed by 5 (the only one originating fromthe Tanpok basin). The best progeny produced the equivalent of1275 kg of dry cocoa per hectare, cumulated over five years,which was similar to the mean for neighbouring trial C0. Popu-lation 12 seemed to be particularly late.l Yield. The yields of the populations or progenies variedconsiderably. The best populations seemed to be 7 and 1, andthe worst 3. Over seven seasons, the best progeny produced anannual mean yield of 1426 kg of dry cocoa per ha, which wouldhave put it in second place in hybrid trial C0 (Lachenaud et al.1994). The best progenies produced yields approaching 3000 kgof dry cocoa per hectare once in full production (Table 5), i.e.more than the best hybrid progenies in C0. It should be notedthat these yields are potential yields, but similar to actual yieldsgiven the good overall performance of Guianan material inrelation to rot diseases, and the seven blocks studied had per-manent Gliricidia shading, unlike hybrid trial C0 which wasunshaded. In contrast, some progenies seemed to be almost ortotally sterile, with mean yields of 0 to 15 pods per tree over 10years. These low-yielding progenies also showed poor vegetativedevelopment and low yield:vigour ratios (from 0.00 to 0.04). Forinstance, the seven trees of progeny GU 313 had an averagecross-section of 11.8 cm2 in 1998 and did not produce a singlepod in 10 years. This reveals the need for multi-site trials, as themother-tree of this progeny achieved substantial vegetative de-velopment in its wild state, with a height of 20 m.l Average pod weight. When applying a minimum yield of 20healthy pods per tree, average pod weight seemed to be the leastvariable criterion of all those studied. However, the differencesbetween populations were clear and significant, and also con-firmed observations in situ (Lachenaud and Sallée 1993). Popu-lation 1 produced the heaviest pods, followed by 13 and 7. Sometrees , and even one progeny, were found to produce an average

pod weight of over 500 g (up to 600 g), which is high given thatall the healthy pods were taken into account.l Losses caused by rot diseases were negligible at only 1.15%of pods for the trial as a whole. Losses varied from 0 to 3% perpopulation, and from 0.0 to 9.2% per progeny. The highestindividual value was 9.6%. In comparison, overall losses in theC0 trial were 4.7%, with a range of 1.0 to 8.7% per progeny andan individual maximum of 25.0%. Thus Guianan material inthis trial had high overall levels of tolerance to rot diseases.Twenty-four high-yielding trees (more than 200 pods) with rotrates lower than or equal to 1% could be cloned. The populationfrom Tanpok was significantly more susceptible to rot diseasesthan were he Camopi populations.

ConclusionDespite the routine use of molecular markers in recent years,using morphological and agronomic descriptors in genetic di-versity studies is still worthwhile and necessary (Sounigo et al.1997). Indeed, in the absence of QTLs, agronomic descriptorsare still irreplaceable when choosing parents to be incorporatedinto breeding programmes. The wild cocoa trees of FrenchGuiana form a special group and have yet to be used in cocoabreeding. As they have been distributed to numerous countries,it is important to facilitiate their use through characterizationsand assessments accessible to researchers. The agronomic char-acteristics of wild material from the Camopi and Tanpok basinsthat we have just described reveal noteworthy performances ofcertain progenies, or even populations, particularly as regardsyield, the yield:vigour ratio and resistance to rot diseases. Basedon this, we recommend their use in genetic improvementprogrammes, and the practical indications provided by thisstudy (populations and progenies) should assist breeders inselecting breeding material.

AcknowledgementsWe should like to thank Peter Biggins for the translation of theoriginal French text. This study was part of an FIC (FondsInterministériel Caraïbes, France) project associating CIRADand the Cocoa Research Unit (CRU) at the University of theWest Indies (Trinidad and Tobago).

ReferencesBerthaud, J. 1986. Les ressources génétiques pour l’amélioration

Table 5. Mean and maximum potential yields from year 6 to year 10 for the first four blocks in the trialand potential yields of progenies GU 244 (very poor), GU 291 (poor), GU 280 (good). The data are in kgof pods per tree and, in brackets, in kg of dry cocoa per hectare

Year 6 (1993) Year 7 (1994) Year 8 (1995) Year 9 (1996) Year 10 (1997)

Mean 2.42 1.51 4.74 2.40 2.95(353) (220) (691) (350) (430)

Max. 10.91 7.69 20.29 11.60 19.92(1591) (1122) (2960) (1692) (2906)

GU 244 0.00 0.00 0.09 0.11 0.51GU 291 0.18 0.25 1.16 0.09 0.40GU 280 6.91 5.58 14.91 6.50 10.37

(1008) (814) (2175) (948) (1513)


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Sounigo, O., F. Bekele, G. Bidaisee, Y. Christopher et R.Umaharan. 1997. Comparison between genetic diversity dataobtained from morphological, biochemical and molecularstudies. Pp. 20-29 in Cocoa Research Unit, Report for 1997.The University of the West Indies, St Augustine, Trinidad.

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Sounigo, O., S. Ramdahin, R. Umaharan and Y. Christopher.1999. Assessing cacao genetic diversity using IE and RAPD

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Plant Genetic Resources Newsletter, 2000, No. 124 7Plant Genetic Resources Newsletter, 2000, No. 124: 7- 12

Interacciones genéticas entre germoplasmasilvestre y cultivado de Lycopersicon spp. conefectos sobre la calidad del fruto de tomateGuillermo Pratta*, Roxana Zorzoli y Liliana A. PicardiConsejo Nacional de Investigaciones Científicas y Técnicas, Consejo de Investigaciones de la Universidad Nacionalde Rosario, Cátedra de Genética – Facultad de Ciencias Agrarias UNR, CC 14 2123 – Zavalla (Santa Fe) – Argentina.Tel/Fax: +54 341 4970080/85; Email: [email protected]

SummaryGenetic interactions affectingtomato fruit quality in wild andcultivated germplasm ofLycopersicon spp.Genetic interactions affecting fruit qual-ity in wild and cultivated germplasm ofthe genus Lycopersicon were evaluated.Plant materials were accessions LA1385of L. esculentum var. cerasiforme andLA722 of L. pimpinellifolium, a genotypewith normal fruit ripening (Platense cul-tivar) and two mutant genotypes withdelayed fruit ripening (nor and rin culti-vars ) of L. esculentum, and the hybridsamong them. Genetic interactions weremeasured by the modifications in thenumber of flowers per cluster and fruitweight, shape, soluble solids content,colour and shelf-life in the hybrid geno-types relative to parental characters. Wildspecies and their hybrids had more flow-ers per cluster and lower fruit weightthan cultivated accessions. The fruitswere also more rounded and had highersoluble solids contents than those of L.esculentum cultivars. Wild species werefound to carry genes that slow fruit rip-ening and prolong shelf-life without im-pairing colour. Such genes from wild spe-cies were expressed by the hybrids, thusindicating that they are dominant overthe respective alleles of the cultivated to-mato. In addition, when wild specieswere crossed with the nor (non-ripeningmutant) cultivar, the detrimental effectsof the mutant on colour were canceledand fruit shelf-life was prolonged.

Key words: Fruit shelf-life, plantbreeding, plant genetic resources,Principal Component Analysis, pro-ductivity, ripening

ResumenInteracciones genéticas entregermoplasma silvestre ycultivado de Lycopersicon spp.con efectos sobre la calidaddel fruto de tomateSe evaluaron interacciones genéticas conefectos sobre la calidad del fruto entregermoplasma silvestre y cultivado delgénero Lycopersicon. Los materiales uti-lizados fueron las accesiones LA1385 deL. esculentum var. cerasiforme y LA722 deL. pimpinellifolium, un genotipo normalpara la madurez del fruto (cultivar Plat-ense) y dos genotipos mutantes cuyosfrutos maduran con demora (cultivaresNor y Rin) de Lycopersicon esculentum,más los híbridos entre ellos. Las interac-ciones genéticas se midieron por mediode las modificaciones ocasionadas en elnúmero de flores por racimo, peso, for-ma, contenido en sólidos solubles, colory vida en estantería de los frutos de loshíbridos con respecto a los de sus pro-genitores. Las especies silvestres y sushíbridos presentaron mayor número deflores por racimo y menor peso, formaesférica y mayor contenido en sólidossolubles de fruto que los cultivares de L.esculentum. En esta experiencia se encon-tró además que el germoplasma silves-tre aporta genes que retardan la ma-durez del fruto, lográndose así una may-or vida en estantería que no está asocia-da a una reducción en el color. Los genesde origen silvestre manifestaron un com-portamiento dominante, ya que se ex-presaron también en los híbridos. Porotra parte, la combinación de estos genesde origen silvestre con el gen nor anulólos efectos desfavorables del mutantesobre el color y prolongó la vida en es-tantería de los frutos.

ARTICLE

RésuméInteractions génétiques chezdes génotypes sauvages etcultivés de Lycopersicon spp.qui présentent des effets sur laqualité du fruit de la tomate.Des interactions génétiques affectant laqualité du fruit parmi des génotypessauvages et cultivés du genreLycopersicon ont été évalué dans ce tra-vail. Les matériaux végétaux utilisés ontété les accessions LA1385 chez L.esculentum var. cerasiforme et LA722 chezL. pimpinellifolium, un génotype normalpour la maturité du fruit (cultivarPlatense) et deux génotipes mutants quila retardent (les cultivars Nor et Rin) chezL. esculentum, plus des hybrides entreeux. Les interactions génétiques ont étémesurées à travers des modificationsoccasionnées dans le nombre de fleurspar racème, le poids, la forme, le contenuen solides solubles, la couleur et la vieaprès la récolte des fruits des hybrides àl´égard de ceux de leurs parents. Lesespèces sauvages et leurs hybrides ontprésenté un nombre plus grand de fleurspar racème et un poids plus bas, uneforme sphérique et un contenu plusgrand de solides solubles du fruit que lescultivars de L. esculentum. En outre, il aété montré dans cette expérience, que lesespèces sauvages apportent des gènesqui retardent la maturité des fruits, enobtenant ainsi une vie plus longue aprèsla récolte qui nést pas associée à uneréduction de la couleur. Les gènesdórigine sauvage ont montré uneconduite dominante car ils se sont aussiexprimés chez les hybrides. Dáilleurs, lacombinaison de ces gènes aves le gènenor a annulé les effets défavorables dumutant sur la couleur et a donné plus depuissance à la prolongation de la vie aprèsla récolte des fruits.

IntroducciónEn los programas de mejoramiento genético de tomate(Lycopersicon esculentum) la calidad del fruto expresada como elpeso, la forma y el contenido en materia es uno de los aspectosmás importantes (Farghaly et al. 1989; Azanza et al. 1995). Estosfactores influyen en la determinación del valor comercial delfruto. El mantenimiento de estas características durante el mayortiempo posible después de la cosecha amplía las posibilidadesde comercialización del producto, especialmente cuando se

destina al mercado en fresco (Mutschler et al. 1992; Kramer yRedenbaugh 1994).

Dentro del acervo genético de la especie se han identificadoalgunos mutantes espontáneos que alteran el proceso demadurez (Stevens 1986; Tigchelaar 1986). Entre ellos seencuentran nor (non-ripening) y rin (ripening inhibitor ), localizadosen los cromosomas diez y cinco, respectivamente. Ambosmutantes han sido descritos como genes recesivos que alteran la


producción del etileno durante la senescencia (Robinson y Tomes1968). Los frutos de estos mutantes al estado homocigotapresentan una mayor vida en estantería pero no adquieren colorrojo, lo que reduce su calidad comercial (Tigchelaar et al. 1978).Su utilización en los programa de mejoramiento genético comoprogenitores de materiales de “larga vida en estantería” se velimitada porque aun en la condición heterocigota provocanefectos pleiotrópicos indeseables sobre el color, el pH, el sabor y elaroma (Buescher et al. 1976). Sin embargo, el tipo y la magnitudde dichos efectos dependen del material genético al que sonincorporados (Tigchelaar et al. 1978).

Por otro lado, las especies silvestres de Lycopersiconrepresentan una importante fuente de variabilidad genética(Rick 1979; Hermsen 1984). Los taxones silvestres L. esculentumvar. cerasiforme y L. pimpinellifolium poseen frutos de menor tamañoy peso que los cultivares comerciales pero de alta calidad, siendoademás de fácil cruzamiento con la variedad doméstica (Rick1973). Bajo el supuesto de que en condiciones naturales elmantenimiento de las propiedades organolépticas durante máslargo tiempo podría ser una estrategia adaptativa para atraer alos predadores y asegurar así la dispersión de las semillas,podrían encontrarse valores de interés fitotécnico para el carácter“vida en estantería” de los frutos dentro del germoplasmasilvestre. De acuerdo a esta hipótesis, los genes de origen silvestrecuya acción genética difiera de nor y rin según resultados previos(Pratta et al. 1996; Zorzoli et al. 1998), se convertirían en unaalternativa para el mejoramiento del carácter, evitando los efectosperjudiciales ocasionados por el uso de los mutantesespontáneos de L. esculentum. Al mismo tiempo, una forma decompensar la reducción en el tamaño de sus frutos podría ser elaumento en el número de frutos por planta, manteniendo asíconstante el peso total de frutos producidos por planta. Lasformas silvestres presentan un mayor número de flores porracimo (Vallejo et al. 1994), carácter que podría ser interpretadocomo un indicador de la productividad potencial.

El objetivo del trabajo aquí presentado fue evaluar lasmodificaciones en caracteres de productividad y de calidad delos frutos, especialmente las referidas a la madurez, producidaspor la interacción genética entre germoplasma silvestre ycultivado del género Lycopersicon .

Material y métodosSe utilizaron los siguientes genotipos: de L. esculentum: cultivaresNor (homocigota recesivo para el locus nor y homocigotadominante para el locus rin, nor nor/rin+rin+), Rin (homocigotadominante para el locus nor y homocigota recesivo para el locusrin, nor+nor+/rin rin), Platense (homocigota dominante para losloci nor y rin, nor+nor+/rin+rin+; representa el genoma normal dela especie) y Tommy (híbrido comercial de larga vida de genotipodesconocido para ambos loci que fue el testigo de estaexperiencia); de L. esculentum var. cerasiforme: accesión LA1385,procedente del Tomato Genetics Resources Center, Departmentof Vegetable Crops, University of California at Davis, CA, USAy originaria de Quincemil, Cusco, Perú; de L. pimpinellifolium:accesión LA722, procedente del Tomato Genetics ResourcesCenter, Department of Vegetable Crops, University of Californiaat Davis, CA, USA y originaria de Trujillo, La Libertad, Perú;

híbridos: F1 (Nor x Rin), F1 (Nor x Platense), F1 (Nor x LA1385) yF1 (Nor x LA722). Las constituciones genéticas de LA1385 yLA722 para los loci nor y rin son desconocidas. En cuanto a loshíbridos, se utilizó siempre como progenitor femenino el cultivarNor en combinación con los otros genotipos como polinizadoresdebido a que los efectos del mutante nor al estado heterocigotaserían más pronunciados que los de rin.

Los caracteres evaluados fueron: a) de rendimiento: 1-número de flores por racimo (FC, medido a los 80 días de lasiembra), 2- peso del fruto a la cosecha (P, en g), 3- forma delfruto (H/D, cociente entre la altura y el diámetro); b) de calidadde fruto: 4- contenido de sólidos solubles (SS, en°Brix, porcentajede glucosa más fructosa del jugo homogeneizado medido conun refractómetro manual tipo Erma A y rango de medición de 0a 32%), 5- color (porcentaje de reflectancia L que indica laintensidad del color y cociente a/b, en donde “a” es laabsorbencia a 540 nm y “b” a 675 nm, medido con uncromámetro estándar CR-100 como promedio de tresdeterminaciones por fruto en el estado maduro), 6- vida enestantería de los frutos (VE, número de días transcurridos desdela cosecha hasta el inicio de pérdida de turgencia de las paredesdel fruto, evaluada en forma manual). Para la medición de VE,los frutos permanecieron en una habitación climatizada a unatemperatura de 28 ± 3°C y humedad constante. Todos loscaracteres de calidad (con excepción del color) fueron evaluadosen frutos de aproximadamente 45 días pos-antesis.

Los ensayos se realizaron en la Sección Horticultura delCampo Experimental José Villarino (ubicado en la localidad deZavalla, Santa Fé, Argentina, a 33° de latitud sur y 61° de longitudoeste) de octubre a marzo, durante la época normal de siembrapara el cultivo. El diseño fue completamente aleatorizado. Cadaplanta (N = 83) constituyó la unidad experimental para el análisisdel número de flores por racimo. Cada fruto (N = 755, entre seis ydiez frutos por planta) constituyó la unidad experimental para elanálisis de los caracteres de calidad. Los valores medios de cadavariable se compararon mediante el Test de Duncan. Debido a lafalta de normalidad, las variables peso y vida en estantería de losfrutos fueron transformadas mediante una función logarítmica(Snedecor 1964). A fin de estimar las acciones génicas involucradasen la determinación de los caracteres evaluados, se formaroncuatro grupos compuestos por los híbridos F1 (Nor x Rin), F1 (Norx Platense), F1 (Nor x LA1385) y F1 (Nor x LA722), más losprogenitores respectivos. Dentro de cada grupo se calcularon losgrados de dominancia para cada carácter de acuerdo a Mather yJinks (1977). Los grados de dominancia se obtuvieron a partir delcociente d/a, en el que d es el valor genotípico del híbrido (calculadocomo la diferencia entre su media y el promedio entre ambosprogenitores) y a es el valor genotípico del progenitor que presentóel mayor valor para el carácter bajo estudio (calculado como ladiferencia entre la media de dicho progenitor y el promedio entreambos progenitores). Se aplicó el Análisis Multivariado deComponentes Principales (Chatfield y Collins 1986) para clasificarlos genotipos de acuerdo a los caracteres evaluados.

ResultadosEn la Tabla 1 se presentan los valores promedio por genotipo decada variable. Los mayores valores de FC se encontraron en los


materiales silvestres y sus cruzamientos. Las diferencias entrelos híbridos (Nor x LA1385) y (Nor x LA722) no fueronsignificativas. Los cultivares Rin, Tommy y los híbridos entrecultivares de L. esculentum var. esculentum mostraron valoresintermedios, correspondiendo el mínimo a Nor y Platense. Losvalores de P se ordenaron de manera inversa a los de FC,presentando el valor máximo el cultivar Platense y el mínimo laaccesión de L. pimpinellifolium. Sin embargo, para esta variable lasdiferencias entre los híbridos (Nor x LA1385) y (Nor x LA722)resultaron significativas (p < 0.05). Para H/D, el mayor valorcorrespondió a la F1 (Nor x LA1385), seguido de la F1 (Nor xLA722). Los materiales de la forma doméstica mostraron valoresmás bajos de esta variable, a excepción del correspondiente alcultivar Rin, que resultó similar al de LA1385. Los taxonessilvestres también manifestaron los mayores valores de SS. Sushíbridos presentaron valores más elevados respecto a losgenotipos de la forma cultivada de L. esculentum. Los cultivaresRin, Nor, el híbrido entre ellos y Tommy presentaron valoresintermedios, correspondiendo el mínimo al cultivar Platense y laF1 (Nor x Platense). Con relación al color, la formas silvestresmostraron en general los menores valores de L y los mayoresvalores de a/b (Fotos 1, 2 y 3). La vida en estantería de los

Tabl

a 1.

Val

ores

pro

med

io, e

rror

est

ánda

r de

la m

edia

de

los

cara

cter

es a

naliz

ados

(1) p

or g

rupo

de

geno

tipos

(2) y

Tes

t de

Dun

can(3

) . D

atos

tom

ados

en

Zava

lla, S

anta

Fé,

Arg

entin

a, e

n el

per

íodo

199

8/19

99.

Gru

po 1

Gru

po 2

Gru

po 3

Gru

po 4

Test

igo

Nor

(N x

R)

Rin

Nor

(N x

P)

Pla

tens

eN

or(N

x C

)L

A13

85N

or(N

x P

i)LA

722

Tom

my

FC6.

0±0.

2f

9.0±

0.2

cd8.

1±0.

1de

6.0±

0.2

f6.

5±0.

1ef

5.0±

0.2

f6.

0±0.

2f

10.6

±0.3

bc11

.3±0

.2b

6.0±

0.2

f10

.5±0

.1bc

15.8

±0.4

a6.

4±0.

1ef

P47

.4±0

.6d

90.5

±1.1

b59

.1±1

.1d

47.4

±0.6

d74

.8±1

.8c

145.

8±2.

2a

47.4

±0.6

d16

.6±

0.1

e4.

7±0.

0g

47.4

±0.6

d5.

5±0.

0f

1.0±

0.0

h87

.0±1

.0b

H/D

0.8±

0.0

c0.

7± 0

.0d

0.9±

0.0

b0.

8± 0

.0c

0.7±

0.0

d0.

6±0.

0e

0.8±

0.0

c1.

0± 0

.0a

0.9±

0.0

b0.

8± 0

.0c

0.9±

0.0

ab0.

9±0.

0ab

0.8±

0.0

c

SS

5.6±

0.1

de5.

3±0.

0de

5.9±

0.0

d5.

6±0.

1de

4.8±

0.1

e4.

8± 0

.0e

5.6±

0.1

de6.

8±0.

0c

8.1±

0.0

b5.

6±0.

1de

7.4±

0.0

bc9.

0±0.

1a

5.9±

0.1

d

L41

.8±1

.3c

37.4

±0.0

cd57

.2±0

.5a

41.8

±1.3

c41

.6±0

.3c

39.5

±1.2

cd41

.8±1

.3c

38.1

±0.3

cd35

.1±0

.4d

41.8

±1.3

c39

.1±0

.1cd

39.5

±0.1

cd48

.8±0

.0b

a/b

0.4±

0.1

c1.

2±0.

0ab

-0.1

±0.0

d0.

4±0.

1c

1.0±

0.0

b1.

1±0.

0b

0.4±

0.1

c1.

2±0.

0ab

1.3±

0.0

ab0.

4±0.

1c

1.5±

0.0

a1.

4±0.

0a

0.6±

0.0

c

VE

22.7

±0.5

ab18

.1±0

.3bc

d32

.8±0

.8a

22.7

±0.5

ab14

.3±0

.2de

11.4

±0.4

e22

.7±0

.5ab

18.7

±0.1

bc14

.8±0

.1de

22.7

±0.5

ab19

.7±0

.1bc

16.2

±0.1

cde

16.4

±0.3

cde

(1) V

er d

escr

ipci

ón d

e ca

ract

eres

en

MA

TER

IAL

Y M

ÉTO

DO

S.

(2) G

enot

ipos

híb

ridos

: (N

x R

): F 1(

Nor

x R

in),

(N x

P):

F 1 (N

or x

Pla

tens

e), (

N x

C):

F 1 (N

or x

LA

1385

), (N

x P

i): F

1 (N

or x

LA

722)

.(3

) Let

ras

dist

inta

s in

dica

n di

fere

ncia

s si

gnifi

cativ

as a

l 5%

.

Foto 1. (A) Cultivares de Lycopersicon esculentum var.esculentum: Nor, Tommy, Caimanta; (B) L. esculentum var.cerasiforme: LA1385; (C) L. pimpinellifolium: LA722.

Foto 2. Híbridos entre cultivares de Lycopersiconesculentum var. esculentum: F1 (Nor x Rin).


mutantes nor y rin fue superior a la del resto de los genotipos,siendo significativas (p < 0.05) las diferencias entre ellos. Cabedestacar que en segundo término se ubicaron los híbridos conlos taxones silvestres, que presentaron valores aun mayores a losdel cruzamiento entre Nor y Rin. El valor mínimo correspondióal cultivar de madurez normal Platense.

Los grados de dominancia (d/a) para todos los caracteresanalizados por grupo de genotipos (cada genotipo híbrido y susprogenitores) se presentan en la Tabla 2.

En el Análisis Multivariado de Componentes Principales,las dos primeras componentes (o direcciones de mayorvariabilidad CP1 y CP2) explicaron el 91% de la variabilidad totaldel conjunto de genotipos (Tabla 3). CP1 representó el 54% de lavariabilidad total, presentando una estrecha correlación positivacon FC, H/D, SS y a/b, y negativa con P y L. Por otra parte, VE,L y a/b aportaron más a la constitución de CP2, que absorbió un37% de la variabilidad total. En la Figura 1 se muestra ladistribución de los genotipos en el plano determinado por CP1 yCP2. Se observa una clara separación entre genotipos enel eje de CP1 según FC, H/D, SS, a/b, P y L. Estadirección, sin embargo, no permitió discriminar segúnVE, lo que sí se logró en la dirección de CP2.

DiscusiónLos valores encontrados en los diferentes híbridos(Tabla 1) confirmarían que el tipo y la magnitud de lasmodificaciones producidas por efecto del mutante nordependen de las interacciones que se establecen entrelos diferentes genomas en que dicho gen es incorporado(Tigchelaar et al. 1978; Zorzoli et al. 1998). Esto también secomprueba cuando se analizan los grados dedominancia por grupo de genotipos (Tabla 2).

Respecto del peso, Weller et al. (1988) mencionaron laexistencia de poligenes con efectos dominantes en lostaxones silvestres que ocasionarían la reducciónobservada en el peso de los frutos de los híbridos con L.esculentum var. cerasiforme y L. pimpinellifolium (Tabla 1).Los grados de dominancia indicaron acciones génicasde dominancia parcial hacia las formas silvestres,

originando un fruto de menor tamaño (Tabla 2). Si bien estefactor podría ser en parte compensado por una mayor

Tabla 3. Componentes principales (CP), autovalor (A)y proporciones de variancia explicada (Ve) yacumulada (Va) de los caracteres analizados(1)(2)

.

CP1 CP2

FC 0.48 (0.93) 0.05 (0.08)P -0.46 (-0.90) -0.21 (-0.33)H/D 0.39 (0.76) 0.37 (0.59)SS 0.48 (0.95) 0.09 (0.14)L -0.25 (-0.49) 0.50 (0.97)a/b 0.33 (0.64) -0.46 (-0.73)VE -0.06 (-0.11) 0.59 (0.94)A 3.81 2.53Ve 0.54 0.37Va 0.54 0.91

(1) Ver descripción de caracteres en MATERIAL Y MÉTODOS.(2) Los valores entre paréntesis corresponden al coeficientede correlación entre la CP y cada carácter analizado.

Tabla 2. Grados de dominancia(1) para los caracteresanalizados(2) en cada grupo de genotipos(3).

Carácter Grupo 1 Grupo 2 Grupo 3 Grupo 4

FC 1.84 ∞ 0.75 0P 6.34 -0.44 -0.44 -0.81H/D -2.78 0 3 1SS -1 1 0 0L -1.57 -1 0 -1a/b 4.54 1 1 1VE -1.9 -0.49 0 0

(1) Ver forma de cálculo en MATERIAL Y MÉTODOS.(2) Ver descripción de caracteres en MATERIAL Y MÉTODOS.(3) Grupo 1: cv. Nor, F1 (Nor x Rin), cv. Rin; Grupo 2: cv. Nor, F1(Nor x Platense), cv. Platense; Grupo 3: cv. Nor, F1 (Nor xLA1385), LA1385; Grupo 4: cv. Nor, F1 (Nor x LA722), LA722.

-3

-2

-1

0

1

2

3

4

5

-3 -2 -1 0 1 2 3 4

CP1 (54% de la variabilidad total)

CP2

(37

% d

e la

var

iabi

lida

d to

tal)

LA 722

LA 1385

F1 (Nor x LA 722)F1 (Nor x LA 1385)

F1 (Nor x Rin)

F1 (Nor x Platense)

cv. Platense

cv. Tommy

cv. Nor

cv. Rin

Figura 1. Posición de los genotipos analizados en el plano definidopor la Componente Principal 1 (CP1) y la Componente Principal 2(CP2). Los valores por genotipo de las componentes principales CP1y CP2 se obtienen de la suma algebraica de los productos entre losfactores presentados en la Tabla 3 para cada carácter y el valormedio de cada genotipo para ese carácter, respectivamente.

Foto 3. Híbridos entre cultivares de L. esculentum var.esculentum y los taxones silvestres: F1 (Nor x LA1385) y F1(Nor x LA722).


producción de flores por inflorescencia en estos materiales (Tabla1), la incorporación del germoplasma silvestre requeriría variasgeneraciones de selección direccional positiva para recuperar eltamaño de fruto.

Los frutos de los materiales silvestres y los mutantespresentaron forma esférica (H/D es más próximo a uno) adiferencia de los cultivares Platense, Tommy y los híbridos dentrola variedad doméstica, en los que la altura del fruto es menor queel diámetro (Tabla 1). Sin embargo, este hecho es de escasaimportancia para determinar la aptitud comercial del fruto.

Los valores encontrados para sólidos solubles concuerdancon los presentados por Rick (1979) y Weller et al. (1988) (Tabla1). No obstante, existiría una elevada correlación negativa entrepeso del fruto y este carácter (Stevens 1986), ya que un mayorpeso se debería a una mayor cantidad de agua en el fruto,manteniéndose aproximadamente constante el contenido desólidos solubles. Por lo tanto, para incrementar el contenido desólidos solubles sin afectar el rendimiento del cultivo seríanecesario ejercer una fuerte presión de selección sobre amboscaracteres. Asimismo, la correlación positiva entre contenido desólidos solubles y forma, mencionada por Goldenberg y Pahlen(1966) y sugerida por los resultados encontrados en estaexperiencia (Tabla 1), indica que los frutos redondos tienden apresentar un mayor porcentaje de materia seca. Esto podría serdebido a los marcados efectos del germoplasma silvestre sobreambos caracteres.

Teniendo en cuenta que valores más altos de L indicanmenor intensidad del color rojo de los frutos y que el cociente a/b aumenta a medida que madura el fruto, en esta experiencia sedetectaron deficiencias de color en los mutantes de larga vida dela forma cultivada (Tabla 1). Esto no ocurriría en el caso delhíbrido F1 (Nor x Platense) y ni siquiera en la F1 (Nor x Rin),debido posiblemente al carácter recesivo de ambos mutantes.Por tratarse de genes recesivos, el fenotipo del doble heterocigotanor+/nor rin+/rin se asemeja más a un fruto normal que acualquiera de los progenitores. Así, los grados de dominanciaencontrados en el Grupo 2 para L y a/b indican accionesdominantes del genotipo cultivado de L. esculentum var. esculentumsobre el mutante nor mientras que los correspondientes al Grupo1 para las mismas variables sugieren acciones génicas desobredominancia (Tabla 2). En el caso de los taxones silvestres ysus cruzas (Tabla 1), el rango de variación de L y a/b es similaral del cultivar Platense (Mutschler et al. 1992), por lo que su colorno difiere del genoma normal de la forma cultivada. Tambiénpara este carácter los resultados obtenidos sugieren la existenciade acciones de dominancia del genoma silvestre sobre elcultivado (Tabla 2).

Para la vida en estantería, los mayores valores de las formassilvestres respecto al cultivar Platense - pero menores a los de losmutantes (Tabla 1) - confirman resultados de experienciasprevias (Pratta et al. 1996; Zorzoli et al. 1998). Se debe destacar queen el ordenamiento general, los híbridos entre los mutantes delarga vida con L. esculentum var. cerasiforme y L. pimpinellifolium seubican muy próximos a las líneas homocigotas para losmutantes. Los grados de dominancia (Tabla 2) indican accionesgénicas de sobredominancia para el Grupo 1, puesto que, comofuera señalado previamente, el fenotipo de doble heterocigota

tiende al de un fruto normal. En el Grupo 2 se encontródominancia parcial hacia el cultivar Platense, lo quecorrespondería a las interacciones diferenciales que se establecenentre el mutante nor (descripto como recesivo) y los diferentesmateriales genéticos de L. esculentum (Tigchelaar et al. 1978; Zorzoliet al. 1998). Las acciones génicas de dominancia incompleta enlos Grupos 3 y 4 (d/a = 0; Tabla 2) confirmarían la hipótesis de laexistencia de loci génicos en los taxones silvestres del género queprolongarían la vida en estantería de los frutos. Dichos lociserían dominantes sobre el genoma cultivado de L. esculentum einteractuarían aditivamente con el locus nor, causando el efectode potenciación observado en el carácter al comparar las F1 (Norx LA1385) y (Nor x LA722) con las F1 (Nor x Rin) y (Nor xPlatense) (Tabla 1). En consecuencia, sería posible usar elgermoplasma silvestre como progenitor de híbridos de larga vidaen estantería en reemplazo de los mutantes espontáneos de laespecie cultivada y de la aplicación de técnicas biotecnológicas(Hobson y Grierson 1993; Kramer y Redembaugh 1994) queimplican un mayor costo y un cierto grado de resistencia en elconsumidor frente a la adquisición de alimentos genéticamentemanipulados (Imanishi 1988).

Las observaciones mencionadas en los párrafos anteriores secompendian en el Análisis de Componentes Principales.Destacan en la Figura 1 los efectos dominantes del germoplasmasilvestre, cuyos híbridos se encuentran muy cercanos a lasaccesiones de L. esculentum var. cerasiforme y L. pimpinellifolium.Además, es posible observar en dicha figura el carácter recesivode los genes mutantes nor y rin, pues la F1 (Nor x Platense) y la F1(Nor x Rin) se hallan próximas entre sí y al cultivar Platense.También se ponen de manifiesto las acciones génicas desobredominancia que caracterizaron al Grupo 1 a lo largo de laexperiencia, dado que el híbrido entre los mutantes se ubicafuera del rango de variación determinado por sus progenitores.La F1 (Nor x Platense) se encuentra prácticamente en el puntomedio del rango de variación definido por la posición de susprogenitores, lo que concuerda con las acciones génicas dedominancia parcial de los cruzamientos intervarietales entregenotipos normales y mutantes propuestas por Milkova (1976).

Los resultados de esta experiencia confirman en primerlugar los efectos pleiotrópicos sobre los caracteres asociados a lamadurez de los genes mutantes nor y rin al estado homocigota encomparación con los frutos normales de tomate (Buescher et al.1976; Ng y Tigchelaar 1977; Tigchelaar et al. 1978; Pratta et al.1996; Zorzoli et al. 1998). En segundo lugar, señalan laimportancia de considerar al germoplasma silvestre deLycopersicon como fuente de genes para incrementar lavariabilidad en el número de flores por inflorescencia, peso,forma, contenido en materia seca y color (Rick 1973; Lindhout etal. 1991; Vallejo Cabrera et al. 1994), y principalmente parademorar la maduración de los frutos, que en este trabajo estárepresentada por la vida en estantería.

AgradecimientosA los Ing. Agr. Stella Maris García, Inés Teresa Firpo, RicardoMurray y Alejandra Yomi, al Ing. Qco. Enzo Tossi, al Dr. CharlesRick, a la Prof. de francés Mabel B. de Pratta y al Técnico enÓptica Marcelo J. Fusi.


BibliografíaAzanza, F., D. Kim, S.D. Tanksley y J.A. Juvik, 1995. Genes from

Lycopersicon chmielewskii affecting tomato quality during fruitripening. Theor. Appl. Genet. 91: 495-504.

Buescher, R.W., W.A. Sistrunk, E.C. Tigchelaar y T.J. Ng 1976.Softening, pectolytic activity and storage life of rin and nortomato hybrids. HortScience 11: 603-605.

Chatfield, C. y A.J. Collins, 1986. Introduction to MultivariateAnalysis. Chapman and Hall. Londres.

Farghaly, M.A., H.A. Hussein y A.M. Damary 1989. Qualitycriteria of tomato fruit according to cultivar and stage of fruitripening. Assiut Journal of Agricultural Sciences 20(4): 97-107.

Goldenberg, J.B. y A. Pahlen 1966. Genetic and phenotypic corre-lation between weight and dry matter content of tomatofruits and their heritabilities. Bol. Genético 2:1-15 (Argen-tina).

Grierson, D. y R. Fray 1994. Control of ripening in transgenictomatoes. Euphytica 79:251-263.

Hermsen, J.G. 1984. Some fundamental considerations on inter-specific hybridization. Iowa State Journal of Research 58(4):461-474.

Hobson, G. y D. Grierson 1993. Tomato. En: Biochemistry of fruitripening. Ed. por G. Seymour; J. Taylor y G. Tucker. Págs.405-442.

Imanishi, S. 1988. Efficient ovule culture for the hybridization ofLycopersicon esculentum and L. peruvianum , L. glandulosum .Japan. J. Breed. 38(1): 1-9.

Kramer, M.G. y K. Redenbaugh 1994. Commercialization of atomato with an antisense polygalacturonase gene: TheFLAVR SAVRTM tomato story. Euphytica 79:293-297.

Lindhout, P., G. Pet, R. Jansen y H. Jansen 1991. Genetic differ-ences in growth within and between Lycopersicon species.Euphytica 57:259-265.

Mather, K. y J.E. Jinks 1977. Introduction to biometrical genetics.Chapman and Hall. Londres.

Milkova, L. 1976. Combining ability for soluble solids in a tomatodiallel cross. Genet. Agric. 30: 327-334.

Mutschler, M.A., D.W. Wolfe, E.D. Cobb y K.S. Yourstone 1992.Tomato fruit quality and shelf-life in hybrid heterozygous forthe alc ripening mutants. HortScience 27(4): 352-355.

Ng, T.J. y E.C. Tigchelaar 1977. Action of the non-ripening (nor)mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci.102(4): 504-509.

Poysa, V. 1992. Use of Lycopersicon cheesmanii and L. chmielewskiito increase dry matter content of tomato fruit. Can. J. PlantSci. 73: 273-279.

Pratta, G., R. Zorzoli y L.A. Picardi 1996. Evaluación decaracteres de interés agronómico en especies del géneroLycopersicon. Horticultura Argentina 15(39):25-32 (Argen-tina).

Rick, C.M. 1973. Potential genetic resources in tomato species:clues from observations in native habitats. En: Genes, en-zymes and populations. Ed. Svb, Plenum, New York. Págs.255-269.

Rick, C.M. 1979. Potential improvement of tomatoes by con-trolled introgression of genes from wild species. Proceedingsof the Conference on Broadening Genetic Base Crops,Wagenigen, 1978. Pudoc, Wagenigen. Págs. 167-173.

Robinson, R.W. y M.L. Tomes 1968. Ripening inhibitor: a genewith multiple effects on ripening. Rpt. Tom. Genet. Coop. 6:22-23.

Snedecor, G. 1964. Métodos Estadísticos - 5ta Edición. CompaníaEditorial - México.

Stevens, M.A. 1986. Inheritance of tomato fruit quality compo-nents. En: Plant Breeding Review (4). Ed. por AVI PublishingCo. Págs. 273-311.

Tigchelaar, E.C. 1986. Tomato Breeding. En: M.J. Basset (ed.).Breeding vegetable crops. Westport, CT, USA. AVI Publish-ing Company, Inc. Págs. 135-170.

Tigchelaar, E.C., W.B. Mc Glasson y R.W. Buescher 1978. Geneticregulation of tomato fruit ripening. HortScience 13(5):508-512.

Vallejo Cabrera, F.A., J.H Pava, J.A. Vargas y P.A. Arango Ángel1994. Caracterización morfo-agronómica de especies y

variedades botánicas del género Lycopersicon. ActaAgronómica 44(1/4): 37-50 (Colombia).

Weller, J.I., M. Soller y T. Brody 1988. Linkage analysis of quanti-tative traits in an interspecific cross of tomato (Lycopersiconesculentum x Lycopersicon pimpinellifolium) by means of geneticmarkers. Genetics 118: 329 -339.

Zorzoli, R.; G. Pratta y L.A. Picardi 1998. Efecto de los mutantesnor y rin y de genes de origen silvestre sobre la calidadpostcosecha de los frutos de tomate. Mendeliana 13(1):12-19(Argentina).

Plant Genetic Resources Newsletter, 2000, No. 124 13Plant Genetic Resources Newsletter, 2000, No. 124: 13 - 16

Cultivation and use of African yam bean(Sphenostylis stenocarpa) in the Volta Region of GhanaG.Y.P. Klu 1*, H.M. Amoatey1, D. Bansa1 and F.K. Kumaga2

1 Biotechnology and Nuclear Agriculture Research Institute, Ghana Atomic Energy Commission, PO Box AE50, AtomicEnergy, Accra, Ghana2 Department of Crop Science, University of Ghana, PO Box LG44, Legon, Accra, Ghana

SummaryCultivation and use of Africanyam bean (Sphenostylisstenocarpa) in the Volta RegionThe African yam bean (Sphenostylisstenocarpa Hochst ex A. Rich) is a legumi-nous crop found in the Volta Region ofGhana. Three expeditions were under-taken in the region between September1998 and March 1999 to investigate theextent of cultivation and use of the beanas the basis for research into its develop-ment and promotion as a major crop. Itscultivation is currently localized aroundNkwanta and Ho West Districts. It isgrown as a minor crop in mixed associa-tion with yam and cassava. It is usedextensively in various dietary prepara-tions and has potential for supplement-ing the protein requirements of manyfamilies throughout the year. Its currentlow status as a minor crop means thatthis potential is largely unexploited. Re-search efforts are required to improve itsagronomic characteristics and promoteits cultivation and use as a major crop.

Key words: African yam bean, foodsecurity, Ghana, legume, pulse,Sphenostylis stenocarpa,underexploited crop

ResumenCultivo y uso del frijol ñameafricano (Sphenostylisstenocarpa) en la Región delVolta, GhanaEl frijol ñame africano (Sphenostylis steno-carpa Hochst ex A. Rich) es una legumi-nosa cultivada en la Región del Volta enGhana. Entre septiembre de 1998 y mar-zo de 1999 se emprendieron tres expedi-ciones en la región para investigar la ex-tensión de su cultivo y uso como basepara estudiar su desarrollo y promocióncomo producto importante. El cultivo selocaliza actualmente en torno a los distri-tos de Nkwanta y Ho West . Se consideracomo un producto secundario asociadoal ñame y la mandioca. Se usa extensa-mente en varias recetas y tiene potencialpara cubrir las necesidades de proteínascomplementarias de muchas familias a lolargo del año. Su actual consideracióncomo cultivo secundario significa queeste potencial permanece en gran medi-da inexplotado. Se precisan investiga-ciones para mejorar sus característicasagronómicas y promover su cultivocomo producto agrícola principal.

ARTICLE

RésuméCulture et utilisation du poistubéreux africain (Sphenostylisstenocarpa) dans la Région dela Volta au GhanaLe pois tubéreux africain (Sphenostylisstenocarpa Hochst ex A. Rich) est une es-pèce légumineuse poussant dans la Ré-gion de la Volta au Ghana. Commepremière étape de recherche pour sondéveloppement et sa promotion en tantque culture majeure, trois missions ontété effectuées dans cette région entre sep-tembre 1998 et mars 1999 pour évaluerl’étendue de la culture et l’usage de cepois. Sa culture est actuellement localiséeaux alentours de Nkwanta et des HoWest Districts. Il est cultivé en tant queculture secondaire en association avecl’igname et le manioc. Il est utilisé cour-amment dans de nombreuses prépara-tions culinaires et serait un complémentprotéique potentiel disponible toutel’année pour de nombreuses familles.Son statut actuel de culture mineure in-dique que ce potentiel est largement in-exploité. Des efforts de recherches sontdonc nécessaires pour améliorer ses car-actéristiques agronomiques et promou-voir sa culture et son utilisation en tantque culture majeure.

IntroductionGrain legumes constitute the main source of protein in the dietsof the average Ghanaian home. The most important ones arecowpea (Vigna unguiculata), groundnut (Arachis hypogaea) and limabean (Phaseolus lunatus). However, there are other pulses thatcould help meet dietary needs but are cultivated only in local-ized areas and used less. These underexploited legumes includeAfrican yam bean (Sphenostylis stenocarpa), bambara groundnut(Vigna subterranea) and pigeon pea (Cajanus cajan).

The African yam bean is grown in West Africa, particularlyin Cameroon, Côte d’Ivoire, Ghana, Nigeria and Togo (Porter1992). In Ghana it is found in localized areas in the VoltaRegion, where it is grown by peasant farmers as a security crop.It is in danger of extinction because of the high premium placedon the major legumes listed above and others such as soya bean.An additional problem is that this bean receives no researchattention locally.

The African yam bean is grown for both its edible seeds andits tubers. It is a vigorous vine, which twines and climbs toheights of about 3 m and requires staking. It flowers profusely in100 to 150 days, producing brightly-coloured flowers, which maybe pink, purple or greenish white. The slightly woody pods

contain 20 to 30 seeds, are up to 30 cm long and mature within170 days. The plant produces underground tubers that are usedas food in some parts of Africa and that serve as organs ofperennation in the wild (Duke et al. 1977; Anon 1979; Porter 1992).The proximate composition of raw beans is shown in Table 1.

The studyThe study involved visits to farming villages in the Volta Region,Ghana. Three expeditions were undertaken between September

Table 1. Proximate composition of raw bean seeds(% dry weight)

Nutrient Range

Protein 18.3 - 21.1Carbohydrate 61.6 - 74.1Fat 0.4 - 2.5Ash 2.3 - 3.2Fibre 4.5 - 6.4

Sources: Duke et al. (1977), Watson (1977) and Edem et al.(1990).


1998 and March 1999 to investigate the extent of cultivationand use of the African yam bean. Discussions were held withfarmers, individually or in groups. Visits were made to farmers’fields when these were accessible. At least two farms werevisited in each village visited.

Study areaIn Ghana, the African yam bean is cultivated in the VoltaRegion, which shares its eastern border with the Republic of

Togo. The study area stretches between 6°N and about 9°N andbetween 0° and 1°E (Fig. 1). Altitude ranges from 0 to 855 mabove sea level. Annual rainfall ranges from less than 1000 mmin the south to between 1500 and 2000 mm in the middle andnorthern belts.

Vegetation type ranges from strand and mangrove swampin the extreme south to Guinea savannah woodland and moistsemi-deciduous forest along the length of the study area. Soiltypes also vary. Typically, the south has infertile, clayey soils,

Fig. 1. Map of Ghana, showing the study area.


which are often inundated with floodwaters. The inland soils,however, are mainly poor in organic matter and nutrients butporous and well drained and support dense vegetation in somedistricts. The main food crops cultivated in the area includeyams (Dioscores spp.) cassava (Manihot esculenta), cocoyam(Xanthosoma sagitiffolium), bananas and plantains (Musa spp.),maize (Zea mays), sorghum (Sorghum bicolor), millet (Pennisetumtyphoides), African rice (Oryza glaberrima) and various vegetables.

CultivationThe African yam bean is said to have been introduced intoGhana from Togo in 1958 (Adansi 1975). However, discussionswith farmers during this study indicate that the crop was grownin the study area well before this date. Its use in the preparationof special meals during festivals and celebration of puberty ritesfor girls may date back centuries. However, no wild relatives ofthe crop were found during the study, nor is there any solidevidence, historical or linguistic, to suggest that the African yambean originated here.

Cultivation and use of the African yam bean in this studyarea is localized mainly in the Nkwanta and Ho West Districts.Several landraces are grown. These are identified by differencesin seed-coat colour. Although the farmers expressed no prefer-ence for any particular colour, lighter-coloured types were en-countered more often than dark-coloured or multicoloured ones.Whereas farmers commonly grew cowpea, soya bean andgroundnut in pure stands, African yam bean was always foundin mixed stands, in association with other crops. No fertilizers orpesticides were applied to any of these crops.

In the Nkwanta District African yam bean was intercroppedwith yam and cassava and was cultivated mainly by womenand children, who constitute the main labour force on thefarms. Traditionally, the farmlands are cleared of their vegeta-tive cover and the soil is formed into mounds. Yam is plantedfirst, on top of the mound, followed by two cuttings of cassavastem on the lower contours of the mound, one on either side.African yam bean is planted a month or two later, after themajor crops have established. Usually, 2-3 seeds are planted perhill, on or close to the mound. The bean seedlings climb thecassava stems for support, with some of them eventually reach-ing the live stakes used by the yams. No special care is providedfor the bean. However, it benefits from weed control, which isdone at least twice before the major crops are harvested inSeptember.

Women and children harvest the beans, while men harvestthe major crops. Local farmers have no basis for estimating seedyield as harvesting is done solely on family demand. Usually thelast pods are harvested and threshed between December andJanuary. These seeds are stored in earthenware to provide partof the family’s food during the lean season. Some farmers arecompelled to sell their seed in the local markets to meet familycontingencies. The price is comparable to that obtained forgroundnut or cowpea.

The cultivation and use of the African yam bean in themiddle belt of the study area have declined over the years. Evenso, it is still grown in some localities, particularly aroundGbadzeme, Biakpa, Logba and Taviefe, where it is called ‘Kulege’

or ‘Kutreku’. There is no gender bias in the cultivation of thecrop in this area, where it is also intercropped with yams,cassava and vegetables. The crop is planted in May, after yamand cassava. Two or three bean seeds are planted adjacent toeach hill. No special pattern is followed but care is taken to sitehills close to juvenile trees saved during land clearing to serve aslive stakes. Weeds are controlled by hand weeding, to the benefitof both major and minor crops.

Dry seeds are harvested piecemeal, to meet family demands,except where farmers intended to sell seeds. This is made pos-sible by the fact that dry pods do not shatter easily. The lastharvest may be done in December. Unthreshed pods are oftentied into bundles and hung or stashed above the fireplace in thekitchen for storage. Smoke from the firewood used for cookingrepels storage pests, thereby facilitating long-term storage.

UseThroughout the study area, the African yam bean is grownprimarily for its dry seeds, which are a nutritious pulse. In theNkwanta District, the Konkombas mill the dry seeds into flour,which is processed into a paste with water and some condi-ments. This is then wrapped into plantain leaves and boiled andeaten as ‘turbani’. The flour may also be mixed with cassavaflour and cooked into a paste eaten with soups or sauces. TheChalas, another ethnic group in the Nkwanta District, boil thedry seeds for about three hours, replacing the water intermit-tently. The cooked beans are made into a sauce and eaten with‘gari’, a roasted cassava product. Some of the farmers inter-viewed reported that the water drained after boiling the beansmay be drunk by lactating mothers to increase their milk pro-duction.

In the Avatime traditional area of the Ho West District, freshmature seeds are added to soups as a protein supplement, whiledry seeds are roasted and milled into flour, which is processedinto sauces or soups with additional condiments for eating withvarious foods. Ethnic groups around Ho roast the seeds and eatthem with maize. The mature green beans are also boiled in thepods, shelled and eaten. In Taviefe and neighbouring villages itwas noted that goats and sheep feed on the dry pods. The cropalso occupies a niche in the sociocultural lives of the traditionalpeople in this area. It is used in the preparation of special mealsduring the celebration of puberty rites for adolescent girls.

Although there are extensive references to the use of thetuberous roots of African yam bean as a source of carbohydratesin West Africa (Okigbo 1973; Anon 1979; Ezueh 1984; Ene-Obong 1992; Porter 1992), the roots were not used in this way inthe study area. Farmers in the Nkwanta District, noted for yamproduction in Ghana, attach no importance to the bean tubersas their yield compares poorly to that of yam. In the Ho-WestDistrict, also noted for production of many local staples such asyam, cassava, cocoyam and plantain, farmers were not evenaware of the tuber-producing ability of the crop.

Other uses to which the crop is put in the study area areimplicit, as these are not obvious to the farmers. It featuresextensively as an intercrop in the traditional farming systemthroughout the study area. Unknown to the traditional farmers,it may be serving as a rich source of leaf litter for improvement of


soil characteristics. The crop also nodulates profusely and prob-ably has high nitrogen-fixing ability, thereby helping to replen-ish soil nitrogen.

Discussion and conclusionThe African yam bean is cultivated in the middle and northernsectors of the study area. It is underexploited and grown mainlyas a minor crop in mixed cropping systems. Once planted, itreceives little special attention from the farmer but may benefitfrom the general agronomic practices applied on the farm. Insome localities its cultivation is left in the hands of women andchildren. Although cultivation of the crop in the region appearsto have diminished over the years, owing to the popularity ofother legumes such as cowpea, groundnuts and soya beans,several landraces can still be found in the hands of farmers. Thismay be because the African yam bean suffers less pest damagethan the other legumes, both in cultivation and in storage.

Throughout the study area the bean is used mainly as apulse crop. The dry seeds serve as a source of protein in variousfood preparations. The dry seeds are used in the preparation ofspecial meals during the celebration of puberty rites for girls inthe Avatime traditional, giving the crop a special role in thesociocultural lives of the people. Its use as animal feed in somelocalities appears to be of minor importance.

Though the African yam bean currently serves only as asecurity crop, it has the potential to meet year-round proteinrequirements if grown on a large scale. Currently, only limitedquantities are offered for sale in local markets, even though theprice per unit of measure is comparable to that of cowpea orgroundnut. Large-scale production should, therefore, lead toincreases in family income. However, the requirement for trel-lises or stakes to support plants appears to be a major obstacle toincreased cultivation.

Modification of the plant’s architecture, through conven-tional breeding or mutation techniques, to obtain semi-erect orerect types, combined with a shorter growth cycle, would makethe crop more acceptable for commercial-scale cultivation. Im-provement in seed-coat characteristics to reduce cooking timewould increase the crop’s use in foods. Mutation inductioncould generate variability in these characteristics from whichplants with desired traits could be selected for use in developingcommercially acceptable varieties.

AcknowledgementsWe thank Mr A.M. Akrofi and Mr G. Akude, AgriculturalExtension Officers in the Nkwanta District, for their technicalassistance. We are most grateful to the many farmers we cameinto contact with for their invaluable support, especially theirwillingness to share indigenous knowledge with us.

ReferencesAdansi, M.A. 1975. Master register of economic plants (exclud-

ing Cocoa) in Ghana. Bulletin No. 4. Crops Research Institute,CSIS, Ghana. 97 pp.

Anon. 1979. Tropical Legumes: Resources for the future. NAS,Washington, DC, USA. 332 pp.

Anon. 1995. Atlas for Ghana. Macmillan, London, UK. 65 pp.Anon. 1996. ICUC 1996 Annual Report. International Centre for

Underutilized Crops, Southampton, UK. 16 pp.

Duke, J.A., B.B. Okigbo and C.F. Reed. 1997. Sphenostylisstenocarpa (Hochst ex A. Rich) Harms. Trop. Grain Leg. Bull.10:4-6.

Edem, D.O., C.I. Amugo and O.U. Eka. 1990. Chemical compo-sition of yam beans (Sphenostylis stenocarpa). Trop. Sci. 30:59-63.

Ene-Odong, E.E. and F.I. Okoye. 1992. Inter-relationships be-tween yield and yield components in the African yam bean,Sphenostylis stenocarpa (Hochst ex. A. Rich.) Harms. Beitr.Trop. Landwirt. 30(3):283-290.

Ezueh, M.I. 1984. The African yam bean as a crop in Nigeria.World Crops 36(6):199-200.

Porter, D. 1992. Economic botany of Sphenostylis (Leguminosae).Econ. Bot. 46(3): 262-275.

Watson, J.D. 1977. Chemical composition of some less com-monly used legumes in Ghana. Food Chem. 2(4):267-271.


Location of an endophytic Neotyphodium sp. withinvarious leaf tissues of wild barley (Hordeumbrevisubulatum subsp. violaceum)Nadeer N. Youssef1 and Frank M. Dugan2*

1 Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83843, USA2 USDA Agricultural Research Service, Western Regional Plant Introduction Station, 59 Johnson Hall, WSU, Pullman, WA99164-6402, USA

SummaryLocation of an endophyticNeotyphodium sp. within variousleaf tissues of wild barley(Hordeum brevisubulatumsubsp. violaceum )Histological techniques enabled visual-ization of fungal hyphae of Neotyphodiumsp. within coleoptile leaf tissues of seed-lings of wild barley, Hordeum brevisubu-latum subsp. violaceum. Hyphae werepredominantly intercellular and com-monly distributed at low density withinmesophyll tissues; hyphae were also im-mediately adjacent to or just inside sheathcells of the vascular bundle, or in contactwith the inner walls of epidermal cells.Intracellular hyphae were extremelyrare; extensive colonization of vasculartissue was lacking.

Key words: Endophyte, Hordeumbrevisubulatum, Neotyphodium, wildbarley

ResumenUbicación de unaNeotyphodium sp. endofíticaen el interior de varios tejidosfoliáceos de cebada silvestre(Hordeum brevisubulatumsubsp. violaceum)Técnicas histológicas permitieron visual-izar hifas de hongos de Neotyphodiumsp. dentro de tejidos foliáceos coleóptilosde plántulas de cebada silvestre, Hor-deum brevisubulatum subsp. violaceum.Las hifas eran predominantemente in-tercelulares y en general distribuidas conbaja densidad dentro de tejidos mesofíli-cos; las hifas estaban también inmediata-mente adyacentes o justo dentro de célu-las de vaina del haz vascular, o en contac-to con las paredes internas de las célulasepidérmicas. Las hifas intracelulares eranmuy raras; no había una colonizaciónextensiva del tejido vascular.

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RésuméLocalisation d’un Neotyphodiumsp. endophyte dans plusieurstissus foliaires d’orge sauvage(Hordeum brevisubulatumsubsp. violaceum)Des techniques histologiques ont permisla visualisation d’hyphes du champignonNeotyphodium sp. dans les tissus foliairesde coléoptile de graines d’orge sauvage,Hordeum brevisubulatum subsp. violaceum.Les hyphes ont été localisés en majoritédans l’espace intercellulaire et distribuésà faible densité dans les tissus demésophylle ; les hyphes étaient soitimmédiatement adjacents soit juste àl’intérieur des cellules de gaine dufaisceau vasculaire ou en contact avec laparoi interne des cellules épidermales.Les hyphes intracellulaires ont étéextrêmement rares ; il n’a pas été noté decolonisation extensive du tissu vasculaire.

IntroductionClavicipitaceous endophytes are now known to occur in severalspecies of wild barley (Wilson et al. 1991a, 1991c). Presence ofendophytic Neotyphodium sp. (Clavicipitaceae, Hypocreales) in onewild barley (Hordeum brevisubulatum subsp. violaceum (Boiss. & Hohen.)Tzvelev) has been demonstrated to affect resistance to Russianwheat aphid (Clement et al. 1997). Similar deterrence of herbivoryhas been demonstrated in other grasses infected by clavicipitaceousendophytes; the subject has been reviewed (Clement et al. 1994; Clay1996). Interest in interactions of endophyte-infected grasses withinsect herbivores, as well as interest in detecting endophytes inforage grasses, has spurred experimentation with techniques forvisualizing hyphae of clavicipitaceous endophytes in vegetativeand reproductive grass tissues (Clark et al. 1983; Saha et al. 1988;Welty et al. 1986a, 1986b; Wilson et al. 1991b; Hignight et al. 1993;Belanger 1996). The endophyte of H. brevisubulatum has been identi-fied as Neotyphodium on the basis of morphology, endophytic habit,and recent sequence data (James F. White, Jr., pers. comm.). Thelocation of the endophyte in vegetative tissues of H. brevisubulatumhas not been reported in detail. We modified published histologicaltechniques (Johansen 1940) in order to define with precision thespatial location of endophytic hyphae relative to various leaf tis-sues in H. brevisubulatum.

Materials and methodsSeeds of H. brevisubulatum subsp. violaceium (PI 440420, collected

from Kazakhstan in 1977) were surface-disinfested for 90 secwith 0.5% sodium hypochlorite + 8 drops of Tween-20 surfac-tant per 100 ml, planted in plastic 11 x 11x 7 cm boxes (alsodisinfested with sodium hypochlorite) filled with sterile ver-miculite, placed in a clean germinator at a constant temperatureof 20°C with a dark:light photoperiod of 8:16 h, and wateredwith sterile distilled water. Three seedlings were harvested by abasal cut on the first foliage leaf just above the coleoptile whenseedlings reached a height of 35-45 mm at 8 days after germina-tion. The basal 0.5-1.0 cm segments of harvested leaves weredisinfested as above and placed on plates of Difco potato dex-trose agar (PDA, amended with 0.1 g/L each of penicillin G andstreptomycin sulfate from Sigma Chemical Co.) for confirmationof presence of the Neotyphodium endophyte and to check for thepresence of other internal fungi; remaining segments were usedto make stained sections and whole mounts. Plates were incu-bated under ambient laboratory conditions (20-24°C and diffuseflorescent light 10-12 h/day).

Clearing and staining was effected by immersion of leafsegments in Carnoy’s solution (Johansen 1940) for 24 h, rinsingin 70% ethanol for 1 min, followed by staining in acid fuchsin in70% ethanol (Johansen 1940) for 48-72 h. Lengths of stainedleaf tissue were inserted between small blocks of stiff styrofoamto provide support; sections were then cut by hand with a sharprazor blade. Observation of sectioning at 10-20X with a SZ-PT


Olympus Stereomicroscope enabled consistent production ofsuitable sections. Sections and whole mounts were mounted in85% lactic acid, allowed to stand for12-24 h for de-staining,then photographed at 100-1000X via bright field (BF) and/ordifferential interference contrast (DIC) on an Olympus BH-2research microscope. The entire procedure was repeated once.For comparison, leaves from tillers of several mature plants ofthe same accession growing in the greenhouse were similarlystained and sectioned.

Results and discussionWhole mounts (Fig. 1) and transverse sec-tions (Fig. 2) of coleoptile leaves clearlyshowed hyphae to be intercellular withinmesophyll tissues of the leaf blade. Mostsuch hyphae were thinly dispersed assingle strands with about half a dozenvisible in any single transverse leaf sec-tion, but occasionally 3-4 hyphae wouldbe nearly contiguous. Hyphae were occa-sionally seen immediately adjacent to, or(less frequently) just within the bundlesheath (Fig. 3), but only extremely rarelyamong tracheary or sieve elements of thevascular bundle. Sometimes hyphae wereseen to exit tissues and grow externally onthe leaf surface (Figs. 4,5). Only veryrarely were hyphae intracellular. Sectionsplated to PDA consistently produced thecharacteristic, off-white, appressed toslightly flocculent colonies of Neotyphodiumbut no other fungus. Spatial distributionof hyphae within tissues of coleoptileleaves, and the degree of colonization ofvarious tissues, conformed with patternsdiscerned in transverse sections of themature leaves from tillers of greenhouseplants.

Although one must use caution inextrapolating these results to plantsgrown under different conditions, someresults here are interesting variants fromwhat is documented for otherclavicipitaceous endophytes in grasses.Most endophytes not producing symp-toms on the host and formerly classifiedin Acremonium (now in Neotyphodium) areconfined to leaf sheaths, i.e. do not ex-tend into the leaf blade (Bacon and deBattista 1991; Glenn et al. 1996). Accord-ingly, isolation of such fungi into culturefrom vegetative tissue has usually tar-geted leaf sheaths, or the pith of culms,for excision and plating to media or fordiagnostic examination (Bacon 1988; Ba-con et al. 1977). However, the endophyteof wild barley is readily isolated from vir-

tually any excised section of leaf (mature as well as juvenile),and as here demonstrated is thinly but consistently distributedin tissue of the first foliage leaf to emerge from the coleoptile andin mature leaves. Some of the clavicipitaceous fungi whichproduce symptoms (e.g. Epichloë typhina) extensively invade theleaf vascular bundle (White and Morgan-Jones 1996), but noextensive invasion was seen here. Hyphae of E. typhina may alsoinvade intracellulary during the formation of conidiomata, but

Figs. 1–5: Fig. 1. Whole mount of leaf tip (DIC). Hypha (arrows) grows longitudi-nally between walls of mesophyll cells. Fig. 2. Transverse section (BF). Intercellu-lar hyphae (arrows) can be seen within the mesophyll and immediately adjacentto the epidermis. Fig. 3. Transverse section (DIC). Hyphae (arrows) immediatelyadjacent to and just within the vascular bundle. Fig. 4. Transverse section (DIC).Hypha (long arrow) exiting epidermal tissue to the exterior; other hyphae (shortarrows) remain clustered below epidermis. Fig. 5. Transverse section (DIC).Hypha external to tissues (long arrow) and internal below epidermis (short ar-rows). Bar = 10 µm in all figures.


in general clavicipitaceous endophytes grow intercellulary intheir hosts (Bacon and de Battista 1991). The site and extent ofinvasion by clavicipitaceous fungi reflect mechanisms of nutri-ent transfer to the fungal symbiont (Bacon and Siegel 1988;White and Morgan-Jones 1996). Collections of plant germplasmrepresent expanded opportunities for investigation of the occur-rence, character and utilization of endophytic infection (Clem-ent et al. 1994).

ReferencesBacon, C. 1988. Procedure for isolating the endophyte from tall

fescue and screening isolates for ergot alkaloids. Appl.Environ. Microbiol. 54:2615-2618.

Bacon, C.W. and J. de Battista. 1991. Endophytic fungi of grasses.Pp. 231-256 in Handbook of Applied Mycology, Vol. 1: Soilsand Plants (D.K. Arora, B. Rai, K.G. Mukerji and G.R.Knudsen, eds.). Marcel Dekker, NY.

Bacon, C.W. and M.R. Siegel. 1988. Endophyte parasitism of tallfescue. J. Prod. Agric. 1:45-55.

Bacon, C.W., J.K. Porter, J.D. Robbins and E.S. Luttrell. 1977.Epichloe typhina from toxic tall fescue grasses. Appl. Environ.Microbiol. 34:576-581.

Belanger, F.C. 1996. A rapid seedling screening method for deter-mination of fungal endophyte viability. Crop Sci. 36:460-462.

Clark, E.M., J.F. White and R.M. Patterson. 1983. Improvedhistochemical techniques for the detection of Acremoniumcoenophilum in tall fescue and methods of in vitro culture ofthe fungus. J. Microbiol. Methods 1:149-155.

Clay, K. 1996. Interactions among fungal endophytes, grassesand herbivores. Res. Pop. Ecol. 38:191-201.

Clement, S.L., W.J. Kaiser and H. Eichenseer. 1994. Acremoniumendophytes in germplasms of major grasses and their utiliza-tion for insect resistance. Pp. 185-199 in Biotechnology ofEndophytic Fungi of Grasses (C.W. Bacon and J.F. White, Jr.,eds.). CRC Press, Boca Raton, FL.

Clement, S.L., A.D. Wilson, D.G. Lester and C.M. Davitt. 1997.Fungal endophytes of wild barley and their effects on Diuraphisnoxia population development. Entomol. Exp. Appl. 82:275-281.

Glenn, A.E., C.W. Bacon, R. Price and R.T. Hanlin. 1996. Molecu-lar phylogeny of Acremonium and its taxonomic implications.Mycologia 88:369-383.

Hignight, K.W., G.A. Muilenburg and A.J.P. van Wijk. 1993. Aclearing technique for detecting the fungal endophyteAcremonium sp. in grasses. Biotech. & Histochem. 68:87-90.

Johansen, D.A. 1940. Plant Microtechnique. McGraw-Hill, NY,523 pp.

Saha, D.C., M.A. Jackson and J.M. Johnson-Cicalese. 1988. Arapid staining method for detection of endophytic fungi inturf and forage grasses. Phytopathology 78:237-239.

Welty, R.E., M.D. Azevedo and K.L. Cook. 1986a. Detectingviable Acremonium endophytes in leaf sheaths and meristemsof tall fescue and perennial ryegrass. Plant Disease 70:431-435.

Welty, R.E., G.M. Milbrath, D. Faulkenberry, M.D. Azevedo, L.Meek and K. Hall. 1986b. Endophyte detection in tall fescueseed by staining and ELISA. Seed Sci. & Technol. 14:105-116.

White, J.F., Jr. and G. Morgan-Jones. 1996. Morphological andphysiological adaptations of the Balansieae and trends in theevolution of grass endophytes. Pp. 133-154 in EndophyticFungi in Woody Plants: Systematics, Ecology and Evolution(S.C. Redlin and L.M. Carris, eds.). APS Press, St. Paul, MN,US.

Wilson, A.D. S.L. Clement and W.J. Kaiser. 1991a. Endophyticfungi in a Hordeum germplasm collection. Plant Genet. Resour.Newsl. 87:1-4.

Wilson, A.D., S.L. Clement and W.J. Kaiser. 1991b. Survey anddetection of endophytic fungi in Lolium germ plasm by directstaining and aphid assays. Plant Disease 75:169-173.

Wilson, A.D. S.L. Clement W.J. Kaiser and D.G. Lester. 1991c.First report of clavicipitaceous anamorphic endophytes inHordeum species. Plant Disease 75:215.

20 Plant Genetic Resources Newsletter, 2000, No. 124 Plant Genetic Resources Newsletter, 2000, No. 124: 20 - 22

Evaluation and characterisation of sugar canegermplasm accessions for their breeding valuesin NigeriaS. Agboire, A.C. Wada* and M.N. IshaqSugarcane Research Programme, National Cereals Research Institute, Badeggi, Private Mail Bag 8, Bida, Niger State, Nigeria

SummaryEvaluation and characterisationof sugar cane germplasmaccessions for their breedingvaluesThirty local sugar cane (Saccharum spp.)accessions were evaluated and charac-terized for smut (Ustilago scitaminea Syd.)resistance during four years under fieldconditions. The results showed that nineaccessions, BD-07, KN-08, LS-01, OG-07,OY-10, OY-11, OY-16, OY-22 and OY-26,were resistant and had high brix contentand other useful yield related traits. Theirreaction to smut under natural and artifi-cial infection indicated that they are natu-rally adapted to the smut fungus. Theaccessions might be safely grown in ar-eas of low smut inoculum density with-out chemical control. The nine accessionsare recommended for incorporation insugar cane breeding schemes for highyield and smut resistance.

Key words: Breeding, characteriza-tion, disease resistance, evaluation,smut, sugar cane, Nigeria

ResumenEvaluación y caracterización deaccesiones de germoplasma decaña de azúcar por su valor ycontribución al mejoramientoen NigeriaSe evaluaron y caracterizaron treinta ac-cesiones locales de caña de azúcar (Sac-charum spp.) por su resistencia al tizón(Ustilago scitaminea Syd.) durante cuatroaños en condiciones de campo. Los re-sultados mostraron que nueve acce-siones, BD-07, KN-08, LS-01, OG-07, OY-10, OY-11, OY-16, OY-22 y OY-26, eranresistentes y tenían alto contenido enbrixio y otros rasgos útiles para el ren-dimiento. Su reacción al tizón en infec-ción natural y artificial indicaba que estánadaptadas naturalmente al hongo deltizón. Las accesiones podían cultivarsesin problemas en zonas de baja densidadde inóculo de tizón sin recurrir a produc-tos químicos. Se recomiendan las nueveaccesiones para su incorporación enplanes de mejora genética de la caña deazúcar con miras al alto rendimiento y laresistencia al tizón.

RésuméEvaluation et caractérisationd’accessions du germoplasmede canne à sucre pour leursvaleurs en amélioration auNigeriaTrente accessions locales de canne à su-cre (Saccharum spp.) ont été évaluées etcaractérisées pour leur résistance au char-bon (Ustilago scitaminea Syd.) durant qua-tre ans en conditions de champ. Les ré-sultats ont montré que neuf accessionsBD-07, KN-08, LS-01, OG-07, OY-10, OY-11, OY-16, OY-22 et OY-26, sont résis-tantes et possèdent une forte teneur Brixainsi que d’autres caractères de rende-ment utiles. Leurs réactions au charbonen conditions d’infection naturelle et arti-ficielle ont indiqué qu’elles sont naturel-lement adaptées au champignon du char-bon. Ces accessions peuvent croître dansdes zones à faible densité d’inoculum decharbon sans contrôle chimique. Il seraitprofitable d’introduire ces neuf acces-sions dans les programmesd’amélioration de la canne à sucre pourles caractères de rendement et de résis-tance au charbon.

IntroductionSugar cane (Saccharum spp.) is an important food crop of thetropics and subtropics (Sivanesan and Waller 1986). It is culti-vated in about seventy-four countries between 40oN and 32.5oS,encompassing approximately half the globe (Aikulola 1978).

European sailors introduced sugar cane into Nigeria alongthe western and eastern coasts in the fifteenth century. It wasprimarily grown for chewing and for livestock feed (Naidu1987). Current emphasis is, however, on sugar production.Sugar cane accounts for 62% of world sugar production andsugar beet (Beta vulgaris L.) 38% (Naidu 1987; Fry 1997). InNigeria, as in other tropical countries, sugar cane is the majorraw material used for sugar production.

Four sugar estates (Bacita, Numan, Sunti and Lafiagi) inNigeria grow sugar cane on a relatively large scale, while themajority of smallholder farmers grow soft sugar cane (chewingcane) on land holdings of 0.2–0.5 ha (Anon. 1997). Sugar caneis grown on 25–30 000 ha in Nigeria, of which industrial canecovers about 12 000 ha (Akobundu 1987). The major diseaselimiting production of both industrial and chewing sugar canein Nigeria is smut, caused by the pathogen Ustilago scitaminea Syd.(Wada 1997).

Research on sugar cane is still in its infancy in Nigeria, andtherefore establishment of good sources of sugar canegermplasm, of both exotic and local origin, and its

characterisation are of great importance to provide a diversegenetic base for cane improvement. This study was undertakento evaluate and characterize some local germplasm accessions inorder to gauge their commercial and breeding qualities.

Materials and methodsThirty local sugar cane germplasm accessions collected fromdifferent areas of Nigeria were studied. Four collections eachwere made from former Bendel State (now Edo and Delta), Kanoand Ogun, and nine each from Lagos and Oyo States. Caneswere cut into three-budded sets and planted in single row plotsof 5 m x 1 m at the upland sugar cane research field of theNational Cereals Research Institute at Badeggi. The experimen-tal design was a randomized complete block with three repli-cates. Fertilizer was applied at planting (200 kg N, 100 kg P2O5and 180kg K2O/ha) on a sandy loam soil containing 4.6%organic carbon, (OC), 0.07% organic matter (OM), 0.62% totalnitrogen (TN), 0.05 meg/100g exchangeable K and comprising87.8% sand, 11.0% silt and 1.2% clay.

To investigate the reactions of the accessions to smut, three-budded sets with exposed buds were immersed in a smut sporesuspension (4g spores/litre of sterile water at 4 x 106 spores/ml)for one hour as described by Nasr (1977). They were removedand incubated in wet jute gunny bags and kept in the shade for

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14 hours. The sets were then planted in the field the followingmorning. Whip-like smut structures appeared 5 months afterplanting (MAP) and ratooning (MAR).

Data on stalk colour, stalk length, stalk diameter, stalkweight, number of chewable/millable stalks per stool and fieldsucrose (brix) at harvest (12 MAP) were collected from 10 ran-domly selected stalks. Stalk length was measured from groundlevel to the top visible dewlap using a 4-metre rule. Stalk diam-eter was taken at the mid portion of the stalk with a pair ofvernier callipers. The number of chewable/millable stalks wascounted using a tally counter, and field sucrose (brix) wasmeasured using a hand-held refractometer.

The smut reaction data were arcsine transformed accordingto Gomez and Gomez (1984) and were subjected to analysis ofvariance along with the agronomic data. Mean separation wasdone using least significance difference (LSD) at the 5% prob-ability level. The accessions were characterized as recommendedby Hutchinson and Daniels (1971).

Results and discussionIt is important to have large numbers of sugar cane germplasmaccessions in genebanks to allow diverse germplasm to be madeavailable for use in sugar cane improvement programmes(Alexander 1989). From the present study, analysis of variancerevealed significant differences between accessions in yield andyield-related traits (Tables 1 and 2). Five accessions, BD-07, KN-

08, OY-10, OY-16 and OY-26, combined high brix and juicecontent with a desirable number of stalks per stool. Shah et al.(1966) reported that these traits are directly related to cane yield.Other accessions, KN-10, LS-16, LS-17, OG-03 and OY-22, hadprofuse tillering and a high number of stalks per stool.

Nine of the accessions were consistently smut resistant fromthe initial crop through to the second ratoon crop (Table 3). Thissuggests that the accessions are naturally adapted to the smutpathogen. The accessions were accordingly classified intogroups based on their resistance to smut and their usefulnessfor breeding and/or for direct planting by cane farmers wheresmut poses a major problem to sugar cane production (Table 4).

Table 2. Agronomic and quality characteristics of some sugar cane germplasm accessions maintained atBadeggi, Nigeria

Accession Stalk Stalk Stalk Brix Stalk No. of millablecolour length (cm) wt. (kg) diameter (cm) stalks/stool

BD-02 Purple 2.4 0.9 14.0 2.8 11.5BD-03 Purple 2.4 1.1 15.3 2.4 10.5BD-06 Purple 2.1 1.0 16.2 2.9 10.6BD-07 Yellow 3.6 1.8 22.0 3.4 14.3KD-01 Yellow 2.7 1.3 16.3 3.2 14.0KD-10 Green 3.1 1.6 20.6 2.8 12.0KN-06 Green 2.6 1.1 14.8 2.5 11.0KN-08 Purple 1.9 0.9 16.9 3.0 10.8KN-10 Green 1.8 1.0 17.0 3.3 12.5LS-01 Green 1.9 1.2 14.5 3.1 13.1LS-05 Green 2.3 0.7 16.0 2.7 12.7LS-09 Yellow 2.5 0.9 15.3 3.3 13.6LS-15 Green 1.7 1.0 14.6 3.1 10.0LS-16 Green 2.6 1.2 14.3 2.9 13.2LS-17 Green 2.8 1.4 17.0 2.7 11.8LS-20 Yellow 3.0 1.2 16.1 3.2 12.5LS-22 Green 2.1 0.9 17.2 2.5 12.7OG-03 Purple 1.8 1.0 18.5 2.8 13.3OG-11 Brown 2.1 1.2 15.8 3.1 14.0OG-07 Brown 1.7 1.0 20.1 3.0 11.5OG-09 Green 2.3 1.4 16.7 2.6 10.8OY-01 Green 1.9 1.3 14.9 2.8 9.9OY-09 Brown 2.6 1.3 15.6 2.9 10.8OY-10 Purple 3.4 1.9 21.8 3.0 12.8OY-11 Green 1.8 0.9 14.3 3.1 13.6OY-12 Purple 2.4 1.1 17.4 2.5 11.7OY-16 Purple 3.0 1.7 20.8 3.4 13.7OY-18 Purple 2.3 1.0 18.0 2.6 9.8OY-22 Purple 2.0 0.8 16.3 2.8 12.1OY-26 Purple 3.2 1.5 21.7 3.4 13.6

LSD (P = 0.05) 0.21 0.16 1.55 0.16 1.45CV (%) 5.44 8.26 5.55 3.35 7.31

Table 1. Selected terms from the analysis of variancefor yield-related traits and smut reaction of 30 sugarcane accessions grown at Badeggi in Nigeria

Variable MS F P

Stalk length 0.724 42.05 <0.001Stalk weight 0.303 30.71 <0.001Brix 17.336 19.38 <0.001Stalk diameter 0.227 24.00 <0.001Millable stalks/stool 5.705 7.26 <0.001Smut reaction

1993 crop 608.179 46.73 <0.0011st ratoon 1994 447.49 23.42 <0.0012nd ratoon 1995 553.21 32.54 <0.001


AcknowledgementThe authors are grateful to the management of National CerealsResearch Institute, Badeggi for support and permission to pub-lish this work.

ReferencesAikulola, E.O. 1978. The problems of sugarcane farmers in Nige-

ria, Pp. 17-19 in: Proc. Int. Symp. on Sugarcane in Nigeria.NCRI, Badeggi.

Table 3. Reaction of 30 local Nigerian sugar cane accessions to smut disease (%)

Smut reaction (%)

Accession Stalk colour 1993 crop 1st ratoon (1994) 2nd ratoon (1995) Class*

BD-02 Purple 49.47 44.99 48.18 SBD-03 Purple 29.54 36.94 41.00 SBD-06 Purple 26.95 34.88 38.35 SBD-07 Yellow 0.00 15.49 18.85 RKD-06 Yellow 44.90 41.16 45.57 SKN-08 Green 0.00 0.00 0.00 RKN-10 Green 28.48 37.94 36.13 SKD-10 Purple 42.41 35.59 46.16 SLS-01 Green 29.00 29.89 29.98 ILS-08 Green 35.81 37.27 47.29 SLS-05 Green 44.85 46.61 42.09 SLS-09 Yellow 42.34 47.97 49.02 SLS-15 Green 40.46 45.00 51.35 SLS-16 Green 36.22 42.09 42.11 SLS-17 Green 42.29 38.94 43.18 SLS-20 Yellow 37.97 40.01 43.37 SLS-22 Green 41.77 51.91 50.77 SOG-03 Purple 29.90 33.56 36.71 SOG-11 Brown 36.87 38.98 44.00 SOG-07 Brown 24.23 19.70 20.86 ROG-09 Green 51.03 47.45 40.05 SOY-01 Green 39.17 43.87 49.68 SOY-O9 Brown 42.45 50.35 53.74 SOY-10 Purple 14.76 22.73 20.50 ROY-11 Green 19.94 25.26 22.49 IOY-12 Purple 45.00 45.00 49.04 SOY-16 Green 26.83 19.18 19.12 ROY-18 Purple 39.20 50.60 53.76 SOY-22 Purple 15.86 24.56 21.92 ROY-26 Purple 0.00 20.93 23.03 R

LSD (P = 0.05) 5.85 7.09 6.69CV (%) 11.30 12.34 10.88

* R=resistant; S=susceptible; I=intermediate

Table 4. Characterization of Nigerian sugar caneaccessions according to their usefulness forbreeding new varieties

Disease Accessions Significancescore (%) for breeding

0 – 14 BD-07, KN-08, Highly resistantOG-07, OY-10, accessions. Very usefulOY-16, OY-22, in hybridization schemesOY-26 as parents.

15 – 25 LS-O1, OY-11 Intermediate resistance.Usefulness inhybridizationquestionable.

>25 BD-O2, BD-O3, Highly susceptibleKN-10, LS-05, accessions. Not useful inLS-09, LS-15, hybridization.LS-17, OG-07,OY-01, LS-20,KD-06 etc.

Akobundu, I.O. 1987. Sugarcane. Pp. 414-416 in Weed Science inthe Tropics: Principles and Practices (I.O. Akobundu, ed.).John Wiley and Sons, New York.

Alexander, K.C. 1989. Durable resistance to red rot and smut diseasesof sugarcane. Pp. 257-275 in Sugarcane Varietal Improvement(M.K. Naidu, J.V. Screenivasan and M.N. Premachandran, eds.).Sugarcane Breeding Institute, Coimbatore, India.

Anonymous. 1997. Sugarcane Varietal Development and Researchin Nigeria. Paper prepared by NCRI for FMNAR, Abuja. 10 pp.

Fry, J. 1997. A global perspective of the sugar industry. Pp. 1-16in Intensive Sugarcane Production: Meeting the challenge be-yond 2000 (B.A. Keating and J.R. Wilson, eds.). CABI Pub-lishing, Wallingford, UK.

Gomez, K.A. and A.A. Gomez. 1984. Statistical Procedures forAgricultural Research. Second Edition. John Wiley and Sons,New York.

Hutchinson, P.B. and F. Daniels. 1971. A rating scale for sugar-cane characteristics. Pp. 128-131 in Proc. XIVth ISSCT Con-gress, Baton Rouge, Louisiana, USA. ISSCT, Mauritius.

Naidu, K.M. 1987. Potential yield in sugarcane and its realizationthrough variety improvement. Pp. 1-17 in Sugarcane VarietalImprovement (M.K. Naidu, J.V. Screenivasan and M.N.Premachandran, eds.). Sugarcane Breeding Institute,Coimbatore, India.

Nasr, A.I. 1977. Standardization of inoculation techniques forsugarcane smut disease. Sugarcane Path. Newsl. 18:2-5.

Shah, S.S., S. Rajasekaran and R.M. Venkataraman. 1966. Herita-bility of some characters in sugarcane. Indian J. Genet. PlantBreed. 26:107-111.

Sivanesan, A. and J.M. Waller. 1986. Sugarcane diseases. CMI Phyto-pathology Paper No.29. CAB International, Wallingford, UK.

Wada, A.C. 1997. Some important diseases and pests of sugar-cane in Nigeria and their control. Outlook on Agriculture26(2):101–105.


The significance of Vavilov’s scientific expeditionsand ideas for development and use of legumegenetic resourcesB.S. Kurlovich¹*, S.I. Rep’ev², M.V. Petrova², T.V. Buravtseva²,L.T. Kartuzova² and T.A. Voluzneva²¹ Leppälaaksontie 5, as. 1, Pellosniemi 52420, Finland; Mobile tel. +358-405138657² N.I. Vavilov Institute of Plant Industry (VIR), B. Morskaya str. 44, St. Petersburg 190000, Russia.

Tel: +7-812-3144732; Fax: +7-812-3118762; Email: [email protected]

SummaryThe significance of Vavilov’sscientific expeditions andideas for development and useof legume genetic resourcesThis article is a synopsis of the principalexpeditions of N.I. Vavilov and their sig-nificance for scientific research and prac-tice. It also presents a discussion ofVavilov’s law of homologous series inhereditary variation, studies on the prob-lem of the Linnaean species concept, thebotanical and geographical aspects ofplant breeding, and the theory of thecentres of origin of cultivated plants. Thepractical applications of the characteris-tics of accessions of leguminous cropsassembled by Vavilov are described, asis the impact of modern research on de-velopment of Vavilov’s ideas.

Key words: Ecogeographicalapproach, expeditions, geneticresources, legumes, plant breeding,preservation, resistance,transgression

ResumenEl significado de lasexpediciones científicas y lasideas de Vavilov para eldesarrollo y uso de los recursosgenéticos de las leguminosasEste artículo es un resumen de las princi-pales expediciones de N.I. Vavilov y loque significa para la investigación y lapráctica científica. Presenta también unanálisis de la ley de Vavilov de serieshomólogas en la variación hereditaria,estudios sobre el problema del conceptode especie en Linneo, los aspectos botáni-cos y geográficos de la mejora fitogenéti-ca y la teoría de los centros de origen delas plantas cultivadas. Se describen lasaplicaciones prácticas de las característi-cas de las accesiones de leguminosas cul-tivadas reunidas por Vavilov, así comolos efectos de la investigación modernasobre el desarrollo de las ideas deVavilov.

ARTICLE

RésuméImportance des expéditionsscientifiques de Vavilof etperspectives pour ledéveloppement et l’utilisationdes ressources génétiques delégumineusesCet article résume les principalesexpéditions de N.I. Vavilov et leur im-portance pour la recherche scientifiqueet l’expérimentation. Il discute aussi de laloi de Vavilov sur les séries homologuesdans la variation héréditaire, des étudessur le problème du concept d’espèces deLinné, des aspects botaniques etgéographiques de l’amélioration desplantes et de la théorie des centresd’origine des plantes cultivées. Les appli-cations pratiques des caractéristiques desaccessions de légumineuses rassembléespar Vavilov sont décrites ainsi quel’impact de la recherche moderne sur ledéveloppement des idées de Vavilov.

IntroductionNikolai I. Vavilov (1887–1943) is recognized as the foremost plantgeographer, botanist and geneticist of contemporary times. In theearly 20th century, the world did not appreciate the urgency ofprotecting the environment and scientists thought little about thegradual disappearance of valuable plant species. Vavilov wasamong the first to recognize the need for intensive plant collect-ing, research and preservation. He organized and took part inover 100 collecting missions in the major agricultural areas of theworld. During these expeditions, Vavilov paid special attentionto leguminous crops as sources of protein and as means ofincreasing soil fertility (Sinskaja 1991). He considered these issuesto be of the highest priority in biological and agricultural sciencesfor developing sustainable agricultural production.

The books and articles dedicated to the life and scientificactivity of Vavilov (e.g. Sinskaja 1991; Pistorius 1997; Loskutov1999) mention his many research studies on legumes. Manyclose colleagues of Vavilov (e.g. Barulina, Govorov, Zhukovsky)further developed his ideas, selecting legumes as the subject oftheir research. Vavilov and his colleagues revealed a number ofimportant findings in plant biology through such research.However, this vast body of work has not been brought togetherin a single report. We have concentrated our attention on theresults of detailed analysis of legume genetic resources at The

Vavilov Institute of Plant Industry (VIR), named after Vavilovin 1967.

Vavilov’s expeditions and their outcomesVavilov was particularly interested in the sites of ancient agri-cultural civilizations and of mountainous regions. In 1916, hewas sent to Iran and Pamir by the Ministry of Agriculture todetermine the reasons for disease epidemics among the residentRussian garrisons. Vavilov discovered that the wheat used forbread flour was contaminated with seeds of the poisonous grassspecies Lolium temulentum. Vavilov continued his travels in thenorthern and central regions of Iran, in Pamir and other regionsof Central Asia. His main purpose was to collect early varieties ofagricultural plants for testing in northern and droughty areas ofRussia, and to determine the high-altitude limits of agriculture(Vavilov 1987a, 1991). He was also searching for “Persianwheat”, reported to be resistant to many diseases (Bazilevskayaand Bakhareva 1991).

Despite a careful search, Vavilov did not find “Persianwheat” in Iran. However, he did collect many other valuableaccessions, among them leguminous crops including mungbean, chickpea, lentil, everlasting pea, pea, beans and species ofclover, not known at that time in Russia. The materials collected


by Vavilov in Iran and in Pamir formed the foundation of thecollection of leguminous grain crops of the VIR. As a result of hisfirst expedition, 171 grain legumes samples were collected. Sub-sequent collecting missions increased the number of legumeaccessions from Central Asia to 1373 (Table 1). The results of adetailed investigation of these were a starting-point for develop-ing a number of important theoretical and practical generaliza-tions on the origin, geography, genetics, disease resistance andevolution of cultivated plants.

Vavilov’s travels in Iran indicated that there were sources ofancient agriculture in southwest Asia. He also detected particu-lar ecotypes in Pamir, which suggested their origin was in thosemountains. He concluded that southwestern Asia was a centreof origin of a many legume species (Vavilov 1965b). The major-ity of the legumes samples from this centre were small-seededwith specific flower colours determined by recessive genes(Zhukovsky 1971). Cross-pollinated species from southwest Asia(everlasting pea, fodder beans) have a propensity for self-fertili-zation. In the law of homologous series (Vavilov 1920), whichwas largely formulated following the expedition to Iran andPamir, Vavilov noted that variability characterized the entireLeguminosae (Vavilov 1920, 1987b). E.I. Barulina (Vavilov’swife and the principal lentil expert, Fig. 1) established a preciseparallelism in variability of vetch and lentil samples that origi-nated from Iran (Barulina 1930). Their similarity is such thateven an expert finds it difficult to distinguish between seeds ofthese species. This example has become a classic illustration ofVavilov’s law of homologous series in hereditary variability(Sinskaja 1969; Makasheva 1973, 1979; Vavilov 1987a, 1987b).Forms of legumes with various seed and flower colours, obeyingthe law of homologous series, are demonstrated by many speciesof lupin, peas, mung bean, string bean and fodder beans.

In 1924, Vavilov organized an expedition to Afghanistantogether with V.N. Lebedev and D.D. Bukinich (to Herat, Af-ghan Turkestan, Gaimag, Bamian, Hindu Kush, Ba Kafiristan,Jalalabad, Kabul, Kandahar, Baquia, Helmang, Farakh andSehistan). The findings from this expedition supported theconclusions about southwest Asia as a centre of origin of manyplants. It was established that Afghanistan was a major pri-mary focus of formation where there existed a large diversity ofmany major Eurasian crops, representing an inexhaustiblesource of initial material for selection (especially for droughtresistance). Following this hazardous and very fruitful expedi-tion to Afghanistan, Vavilov was awarded the N.M. PrzevalskiGold Medal of the Russian Geographic Society, of which Vavilovwas president from 1931 to 1940.

Vavilov had a special interest in the Khoresm oasis, whoseproximity to Afghanistan and Iran supported the hypothesisthat this territory was also a focus of formation. Vavilov in-spected area around the Amu Darya River (Khiva, Urgench,Gurlen and Tashauz) in 1925. Whereas there were signs in Iranand Afghanistan that cultivated plants had developed locally,Khoresm showed signs of connections with northeast Africaand Egypt that influenced the features of cultivated plants.White-seeded forms of peas, haricot bean and fodder bean werefound in Khoresm, together with particular forms of ground-nuts and alfalfa. Khoresm oasis was characterized by an abun-

dance of many recessive forms of cultivated plants (Vavilov1926; Bazilevskaya and Bakhareva 1991).

In 1921 Vavilov organized a trip to Canada (Ontario) andthe USA. The official purpose was to search for sources ofresistance to drought, necessary for restoring Russian agricul-ture after a severe drought in 1921. Vavilov understood fromhis first visit to North America that the continent was not afocus of intensive formation of plants and agriculture (Vavilov1965b, 1987b). The main foci of plant diversity were to thesouth, in southern Mexico, and Central and South America.Vavilov visited a number of European countries (Britain,France, Germany, Poland, Holland and Sweden) on his returnfrom America in 1922. As a result of these and his subsequenttravels to Germany in 1927, the USA in 1930, Canada andother American countries in 1932–33, he added commercialvarieties and a selection of legumes from many countries to thecollection of VIR.

Vavilov visited Mediterranean countries in 1926: northernAfrica, the islands of Cyprus, Crete, Sicily and Sardinia, andsouthern Europe (Portugal, Spain, France and Greece). He notedthe role of leguminous crops (particularly chickpea) in supplyingboth people and livestock with protein and as a means of increas-ing soil fertility (especially lupin) (Vavilov 1997; Loskutov 1999).He found that the activities of people had little effect on plantdiversity in any of the coastal zones of the Mediterranean. How-ever, a different picture emerged in inland areas, oases and south-

Table 1. Accessions of grain legume crops collectedfrom Central Asia

Crops Quantity of the collected accessions

Personally by As a result ofN.I. Vavilov the consequentin 1916 expeditions

Mung bean 115 680Chickpea 18 434Lentil 16 52Everlasting pea 12 39Pea 8 56Fodder beans 2 112Total 171 1373

Fig. 1. N.I. Vavilov with his wife E.I. Barulina, 1926 (beforetheir expedition to the Mediterranean).


ern slopes of mountains, where the influence of European civiliza-tion was apparent (Vavilov 1997). Vavilov’s special interest inearly agrarian cultures led to an expedition to Syria, Palestine andJordan, where there were traces of ancient agriculture. In Palestinein particular, he found useful forms of white lupins (going underthe local name Tel Karam). These are distinguished by earlymaturity and are now widely used in breeding (Golovchenko et al.1984). Plant collection in Sudan and Ethiopia yielded about 2000accessions of local varieties. In Sudan, Vavilov found valuableforms of white lupin, and in Ethiopia found endemic forms ofeverlasting pea, chickpea, lentil and beans. The flora of Ethiopia isin many respects unique and Vavilov considered it to be anindependent primary Abyssinian focus (Vavilov 1926), and laterthe Abyssinian Centre of diversity (Vavilov 1962).

In 1929 Vavilov organized an expedition to China (Xinjiang–Kashgar, Uch Turfan, Aksu, Kucha, Urumchi, Kulja and Hotan),Japan (Honshu, Kyushu and Hokkaido) and Korea. The purposeof his trip to northern and western China was to determine therole of Central Asia (within the limits of China) in the origin ofcultivated plants of eastern Asia. Returning from northern andwestern China, he inspected the area close to the Chinese borderaround Lake Issyk-Kul and the Syr Darya river basin. Vavilovconcluded from the results of this trip that Central Asia does nothave the features of an independent focus of ancient agriculture:all that was cultivated there had been introduced from the east.His investigations in Japan, however, confirmed it as one of thecentres of cultivated plants (the East Asiatic centre). He foundthat, while Japan had ‘borrowed’ some cultivated plants fromChina, its geographical isolation, its span in latitude, and itsdiversity of climate and ecology generated unique features in thecultivated flora. Wild species and cultivated forms of soybeanwere added to the collection following these expeditions.

Vavilov was interested for a long time in plant geneticresources of Latin America. In 1932, an invitation to the VIInternational Genetic Congress in the USA gave him an oppor-tunity to inspect these regions. Vavilov visited Cuba, Mexico,Ecuador, Peru, Bolivia, Chile, Brazil, Argentina and Uruguayafter the congress. Vavilov detected specific and varietal struc-tures in cultivated plants, and received information about theirhistory, origin and wild relatives. He collected valuable forms ofkidney bean, peanuts and American species of lupin. This wasVavilov’s last overseas trip.

Vavilov also traveled widely in the territory of the formerSoviet Union. He paid special attention to the mountain regions

of the Caucasus and Central Asia, in which there were uniquecultures and wild species, whose role in the creation of culti-vated plants is not doubted. The direct connection of theCaucasus with Iran, Central Asia and Turkmenistan has re-sulted in some similarities with the Southwest Asiatic Centre oforigin of cultivated plants. Vavilov personally participated inexpeditions to more than 50 countries. In 1938–1940, he wroteessays on his journeys under the title “Five Continents”. Theexisting pages of this manuscript were published in 1962 and1987 in Russian (Vavilov 1962, 1987a), and later in English byIPGRI (Vavilov 1997).

The characteristics of the principallegume accessions collected by VavilovMung bean (Vigna radiata (L.) R.Wilczek)Vavilov collected 115 accessions of mung bean during his firstexpedition in 1916. Mung bean is an ancient food crop ofsouthwest Asia. It has a high seed protein content (23.0–32.1%),high lysine, tryptophane and vitamin contents, and goodflavour and cooks quickly (20–40 min). These properties havemade it a favourite food of the local population. Its foliage isuseful as forage for cattle and as a green manure.

Vavilov’s accessions of this crop were studied at the CentralAsian branch of VIR (Popova 1937; Pavlova 1952; Ivanov 1961).From a detailed study of the assembled diversity of mung bean,G.M. Popova developed an intraspecific classification of thiscrop, differentiating three subspecies: Indian (indicus), Chinese(chinensis) and Iranian (iranicus). She also described 63 varietiesof mung bean (Popova 1937). Subspecies iranicus are character-ized by a twisted form of bush, a stalk height of 86-128 cm, 50-180 pods per plant (7-8 cm long), seeds of various colours(yellow, green, grey, brown) and 1000-seed weight of 32.8-49.8g. Most accessions are early maturing, but late-maturing acces-sions also exist. The growing period in Central Asia is 91-122days. The seed yield per plant ranges between 7.3 and 17.0 g(Table 2). Two accessions from Afghanistan (k-2209, k-2216)are highly resistant to drought and are of particular interest.Accessions of Iranian origin differ by having large above-groundbiomass and are potentially useful for creating cultivars in-tended for fodder and green manure.

Chickpea (Cicer arietinum L.)This is an ancient crop of Iran and Central Asia and is repre-sented by numerous local races with small seeds. Plants are

Table 2. Characteristics of mung bean accessions collected by Vavilov in Central Asia in 1916 and 1924

VIR catalogue Origin Growth duration Plant 1000 seed Seed colour Seed mass/No. (days) height (cm) wt. (g) plant (g)

1826 Iran 122 128 40.0 Green 17.02124 Uzbekistan 91 100 41.5 Green 7.32125 Uzbekistan 91 94 49.8 Grey 8.02133 Uzbekistan 91 94 38.2 Dark green 11.02136 Uzbekistan 96 93 45.0 Dark green 12.12164 Uzbekistan 119 100 32.8 Yellow 13.32180 Afghanistan 117 93 46.3 Green 17.22183 Afghanistan 96 86 39.9 Brown 10.02209 Afghanistan 118 88 41.7 Green 10.42216 Afghanistan 91 95.0 39.4 Green 11.7


small and suited only to manual harvesting. However, they arewidely used in breeding. The variety ‘Tadzhiksky 10’ was cre-ated by individual selection from Vavilov’s Tadzhikistansamples. It matures early and is resistant to fusarium wilt. Thisvariety was used to breed the variety ‘Zimistony’, which is alsocharacterized by early maturity and high productivity inTadzhikistan. ‘Milutincky’ was developed from the Iraniansample k-327 by individual selection for Uzbekistan conditions.This cultivar is drought and disease resistant. ‘Tashkent 511’was created from Afghan sample k-223 through mass selection.It has enhanced protein content and disease resistance.‘Kubansky 16’ was developed for conditions of the Kuban re-gion from Uzbek sample k-16. ‘Volga-5’ has shown itself highlyproductive in central Russia. It was bred using accession k-249from Afghanistan.

Lentil (Lens esculenta Moench)Vavilov’s lentil accessions are mainly dwarf forms with smallseeds belonging to a large number of varieties : persica, grisea,violascens and others. There are many endemic forms of early andsemi-early varieties that are drought resistant and have goodcooking properties (Table 3). Pamir sample k-194 matures laterthan all others (growth period 86-102 days). Accessions k-5, k-196, k-434, k-435 have light-blue flowers. Material assembled byVavilov was used as the basis for creating the commercial culti-var ‘Tadzhik 95’, which matures early, has good cooking proper-ties and is drought resistant. ‘Azer’, now cultivated inAzerbaijan, was created using sample k-373 from northern

Afghanistan. It is characteristically tall and has rhomboid beans.Its growth period is 81-95 days and it has a seed protein contentof 29-30% and a 1000-seed weight of 34-37 g.

Pea (Pisum sativum L.)Accessions of pea from Pamir have good winter-hardiness, darkseeds and a specific interaction with Rhizobium bacteria. Manyaccessions of Afghan origin do not produce nodules and havelow nitrogen-fixing ability, even when artificially inoculatedwith Rhizobium. This feature is widely used for control andmatching in genetic research. New varieties of peas with en-hanced biological nitrogen-fixing ability have been bred fromthis material (Tchetkova and Tikhonovich 1986). Currently thereis considerable interest in genetic studies of multimarker linescontaining the sym-locus from a non-nodulating gene of Af-ghan origin.

Pea lines under the common name Mushung (k-181, k-182,k-184 and k-190) were the basis of the commercial cultivar ofwinter peas ‘Mushung mestny’, which was extensively culti-vated in Tadzhikistan. ‘Wostok 55’ was bred for Uzbekistanconditions.

Lupin (Lupinus albus L.)Valuable accessions of white lupin were collected by Vavilovduring his trip to the Mediterranean in 1926 (Table 4). InPalestine, he identified very early, thermally neutral and small-seeded forms of the Jordanian ecotype. In particular, the sampleTel Karam k-290 has a growth period in Ukraine of only 105

Table 3. Characteristics of best lentil accessions collected by Vavilov in Central Asia in 1916

VIR Origin Growth Plant 1000 seed Cooking Droughtcatalogue No. duration (days) height (cm) wt. (g) properties resistance

5 Iran 76-80† 25-30 69-72 Good Average6 Iran 76-80† 25-30 32-42 Good Above average7 Iran 76-80† 35-40 20-25 Good Above average8 Iran 76-80† 25-30 30-43 Good Above average10 Iran 76-80† 35-40 57-70 Average Above average13 Iran 76-80† 25-30 24-29 Good Average14 Iran 76-80† 35-40 56-63 Average Average194 Pamir 76-80† 35-40 34-40 Average Average196 Uzbekistan 86-102‡ 21-31 28-30 Average Average

† Average ripening duration.‡ Late ripening.

Table 4. Characteristics of white lupin accessions (Lupinus albus L.) collected by Vavilov in Palestine andSudan in 1926

Ecotypes Accessions (name, CharactersVIR catalogue no.)

Growth Mass/plant (g) 1000- Seed protein Oil contentduration for seed wt. (g) content (%) (%)spring sowing Green Seed(days) mass mass

Jordanian Tel Karam (k-290) 105 16.2 31.3 300 41.6 10.9Jordanian k-294 111 28.2 33.6 380 42.5 11.5Jordanian k-295 113 34.6 35.0 350 42.8 11.5Jordanian k-298 113 27.5 34.2 340 44.1 11.5Sudanese k-486 140 99.5 40.0 500 41.2 11.3Sudanese k-495 122 82.6 39.5 480 40.5 11.6


days. In contrast, lupin accessions from Sudan are very late, buthighly productive and large-seeded (k-486, k-495). Samples ofthe Jordanian ecotype from Palestine have special value assource material for lupins bred for Russian, Polish and Ukrai-nian conditions. Their use in hybridization with lines of theGeorgian ecotype, following mutagenesis, allowed V.I.Golovchenko to create early and highly productive cultivars,‘Kievsky mutant’, ‘Horizont’ and ‘Druzba’ for Russia and theUkraine (Golovchenko et al. 1984).

Vavilov not only collected and organized research on theassembled material, but also attempted to establish new meth-odologies. He was particularly keen to develop simpler and moreaccessible methods for determining alkaloid content in lupin.This was important in 1928-1929 as von Sengbusch, in Ger-many, had developed such a method but it was kept secret andthe sweet strains of lupin were sold to a private firm. Under themanagement of N.N. Ivanov, and further elaborated by M. I.Smirnova and others, an efficient expression method was devel-oped and adopted in VIR. In 1932, Vavilov wrote the forewordto a work by Ivanov et al. (1932) that was central to the initiationof breeding fodder (sweet) low alkaloid lupin.

Vavilov’s contributions to theory andtheir further developmentVavilov worked on global genetic diversity of cultivated plantsthroughout his life, from which he developed several majortheories that have played an important role in the developmentof botany, genetics and plant breeding. Cornerstones of thiswork include the law of homologous series in hereditary vari-ability, studies on the problem of speciation, differential system-atic-geographical methods for studying crops, botanical andgeographical aspects of plant breeding, and the theory of thecentres of origin of cultivated plants (Vavilov 1920, 1926, 1935,1940, 1997).

Centres of originVavilov’s theory of introduction was based on his understand-ing of the nature of variation of theearth’s vegetation, having identified anumber of areas characterized by par-ticular diversity and richness of speciesand types (Vavilov 1965b). More than70 years ago Vavilov selected five an-cient foci for the origin of cultivatedplants from local flora (Vavilov 1926).Subsequently (Vavilov 1935), he intro-duced methods for better determiningthese foci and identified six foci andtwo centres of origin of cultivatedplants. Vavilov constantly developedand deepened his main thoughts insubsequent publications. He eventuallysettled on seven primary centres of di-versity: Tropical, East Asiatic, South-west Asiatic, Mediterranean, Abyssin-ian, Central American and Andean(Vavilov 1940, 1962, 1987a, 1997) (Fig.

2). He also established specific foci for some of these centres.The localization of centres and foci of origin of cultivated

plants has been further developed by Sinskaja (1969) andZhukovsky (1971) in Russia and by scientists from many othercountries. Sinskaja (1969) revealed broader geographical con-nections and mutual interaction of floras. She distinguishedfive principal areas and updated the list of cultivated crops ineach. Zhukovsky (1971) accepted Vavilov’s definition of centresof diversity but increased the number to 12 and renamed themgene-centres. In our opinion (Kurlovich 1998), there are centresof formation for wild species, where they originated after the lastice-age, and also centres of origin (diversity) for cultivatedplants, where these plants were domesticated.

Vavilov has received considerable support for his determina-tion of centres of diversity (Kurth 1957; Harris 1967; Harlan1971; Brezhnev and Korovina 1981; Mathon 1981). However,some critics believe that it is very difficult to determine the initialgeographical origin of species and prefer the term “centres ofdiversity” instead of Vavilov’s “centres of origin”. Pistorius(1997) considers the term “centres of diversity” safer. Even in amodified form, Vavilov’s theory of centres is used as a theoreti-cal basis for collecting, studying and using genetic resources ofcultivated plants.

Vavilov considered the Mediterranean region and moun-tainous areas of Mexico and the Andes to be centres of origin ofthe genus Lupinus (Vavilov 1926). This has allowed more precisedetermination of the centres of formation and origin (diversity)for specific lupin species (L. albus L., L. luteus L. and L. angustifoliusL.). Our data (Kurlovich 1989), however, indicate that the centreof formation of wild white lupin (L. albus L.) and the primarycentre of origin (diversity) of its initial cultivated forms is theBalkans, where there exists a wide diversity of wild, feral andlocal forms. All three white lupin subspecies (graecus, termis andalbus) are grown in the Balkans. Wild forms with dotted darkbrown seeds and dark blue flowers are found there. The centresof diversity of cultivated white lupins include the Apenninesand Egypt, where cultivated forms of white lupin originated in

Fig. 2. Centres of origin of cultivated plants (Vavilov 1962, 1987a). I – The TropicalCentre, II – The East Asiatic Centre, III – The Southwest Asiatic Centre, IV – TheMediterranean Centre, V – Abyssinian Centre, VI – The Central American Centre,VII – The Andean Centre.


ancient times. In Egypt, forms with pink-and-blue or light pinkflowers exist; in the Apennines, there are forms with greyish-and-light blue or white flowers.

Two close Lupinus species exist in the Pyrenees (L. luteus L.and L. hispanicus Boiss. et Reut.). They have the same number ofchromosomes (2n=52) and there is a wide diversity of wild andcultivated forms of yellow lupin (L. luteus L.) and a long histori-cal record of their growing there. This indicates that thePyrenees was a centre of formation of the wild forms of yellowlupin (L. luteus L.) and the centre of diversity of cultivatedplants (Kurlovich 1998). For similar reasons, we consider thePyrenees to be a centre of diversity for narrow-leafed lupin (L.angustifolius L.).

The agricultural area devoted to lupins has gradually in-creased as a result of varietal development. Consequently, sec-ondary macro and micro centres of diversity of cultivated formshave developed on different continents. These correspond tomany geotypes and ecotypes described by Kurlovich (1998).Secondary centres of diversity of cultivated white lupin occur inBelarus, Chile, France, Germany, Poland and Russia. Secondarycentres of diversity of blue lupin exist in Australia, Belarus,Poland South Africa and southeastern USA. Secondary centresof diversity of yellow lupin occur in Belarus, Germany, Poland,Russia and Ukraine. Results of these studies make it possible, toa certain extent, to provide answers to the most importantquestions of plant introduction and breeding regarding the typeand location of material that should be collected and the pur-pose to which it should be put.

Vavilov’s law of homologous seriesWhen studied under different geographical conditions, anyplant species segregates into a wide range of hereditary forms,which is initially difficult to understand. However, accordingVavilov’s law of homologous series in hereditary variability,intraspecific diversity does have some regularity. The essenceof the law is that closely related species and genera are charac-terized by similar series of variation. Knowing the nature ofsuccession of varieties in one species, one can forecast theexistence of similar forms in other species and genera. Wholeplant families in general are characterized by a definite cycle ofvariability, which exists similarly in all genera and species ofthat family.

Vavilov described variability in the Fabaceae (Leguminosae)and established regularity in its differentiation in separate gen-era and cultivars, by monitoring characters of seeds, fruits, andflowers, and vegetative organs. He analyzed variability of char-acters in accessions of Vicieae, Trifolieae, Loteae, Galegeae andPhaseoleae (Vavilov 1920; 1987b). It became clear that, despitedifferences between these sections, the variability of characterswas similar for all genera within a given family. The genusLupinus is more variable than any other genus of the Fabaceaeand illustrates Vavilov’s law of homologous series in hereditaryvariability (Maissurjan and Atabekova 1974, Kurlovich et al.1995). The law of homologous series in hereditary variabilityindicates which material should be sought and underlies thetheory for the centres of origin of cultivated plants. It alsoindicates where such centres should be located.

Differential systematic-geographical methodsVavilov and his followers used a differential systematic-geo-graphical method to study intraspecific diversity and to deter-mine the centres of origin of cultivated plants (Vavilov 1931).This method consisted of the following:l differentiation of a genus into species and intraspecific di-versity using morphological, hybridisation, cytological and othercharactersl determination of the genotypical composition of a speciesl geographical localization of hereditary forms of a speciesand the centres of their diversity

The large body of theoretical and practical work involved inthese studies allowed Vavilov to comment on the nature androle of species as a system (Vavilov 1931, 1965a). Previously theprevailing ideas were those of Komarov (1931, 1944), based onmonotypic species, according to which species cannot include asystematic unit of a lower rank. The concept of a biologicalspecies based on no crossing between species was also widelyaccepted (Grant 1981, 1984). Vavilov, however, in a study ofseveral hundred species showed the absence of monotypic spe-cies, i.e. species represented by a sole race or form. All the speciesstudied appeared to incorporate a number of forms (genotypes).He considered a species to be a flexible, isolated, complex, mor-phophysiological system linked to a particular environment andarea (Vavilov 1931, 1965a). This led to the understanding of theLinnaean species concept, an integral entity consisting of closelyinterlinked components where the whole and the parts aremerged (Vavilov 1965a; Agaev 1987; Korovona 1987).

Geographical and ecological differentiationVavilov not only paid attention to morphological traits, but alsotook into account geographical and ecological differentiation ofplants. Such an approach based on development of variousintraspecific classifications allowed detailed study of intraspe-cific and varietal diversity of cultivated plants, and develop-ments for their effective use. In the international code for botani-cal nomenclature there exist fixed categories for intraspecificclassification, including subspecies, varieties, sub-varieties andforms.

Vavilov paid particular attention to ecogeographical differ-entiation of a species into ecotypes, genotypes, concultivars etc.(Vavilov 1931, 1965a). For classification purposes, VIR’s scien-tists routinely use anatomical, cytological, paleobotanical, onto-genetic, biochemical, physiological, geographical, genetic andother criteria in addition to the more usual traits. Such a com-prehensive approach is particularly effective for the study ofintraspecific diversity in cultivated leguminous crops. Vavilov’stheories are all interconnected and represent a complex doctrineabout global genetic diversity of cultivated plants. They haveallowed scientists from VIR to develop intraspecific classifica-tions for practically all leguminous crops, particularly peas(Govorov 1937; Makasheva 1979), mung bean (Popova 1937),soya bean (Korsakov 1971; Teplyakova 1997), lupin (Kurlovichand Stankevich 1990), chickpea (Seferova 1997) and vetch.

Continuing Vavilov’s workVavilov’s work in collecting and studying genetic resources of


leguminous plants has been continued by VIR scientists. Thedepartment of leguminous crops of VIR was established in 1924.Vavilov invited his friend L.I. Govorov to manage the depart-ment, and he recruited experts in leguminous plants to the staff.Govorov developed intraspecific classification for peas (Govorov1937), organized breeding work with leguminous crops in theformer USSR and created many pea cultivars. Like Vavilov,Govorov had a tragic destiny; he was arrested and disappeared.

P.M. Zhukovsky was invited to manage the lupin collection.He developed and published important works on interspecificand intraspecific diversity (Zhukovsky 1929). Research on thegenetic resources of lentil was done by E.I. Barulina (Vavilov’swife). She took part in many expeditions, e.g. to Crimea, Georgiaand other regions (Barulina 1930). During the Second WorldWar the department of leguminous crops was managed by N.R.Ivanov, who made a substantial contribution to preserving thecollection during the seige of Leningrad. He carried out impor-tant research on the genetic resources of kidney bean (Ivanov1961). After the Second World War, under the direction ofKorsakov, ecological and geographical research at VIR was modi-fied and databases on genetic resources were created. Korsakovalso contributed to the clarification of centres of origin of varioussoya bean species (Korsakov 1971).

Recent activities at VIRCurrently, VIR has 43 000 accessions belonging to 15 genera and160 species of the Fabaceae, including pea, soya bean, vetch,lupin, faba bean, lentil, everlasting pea, chickpea, cowpea, mungbean and kidney bean. The collection contains genetic resourcesfrom five continents, but most of the accessions were collectedfrom the former USSR and Europe. The staff of the departmentof leguminous crops selects new, valuable lines, works out ad-vanced methods for breeding and performs research on tax-onomy and evolution. Considerable effort is directed at evaluat-ing agronomic traits, including yield factors and tolerance ofbiotic and abiotic stresses, and at ecogeographic research invarious regions of Russia. For the first time in Russia, selectionfor new characters in peas is beginning to produce stipulate

lines with non-dehiscent pods, polyembryonic types and deter-minate types. During recent years, experiments were conductedto determine resistance in pea to Aphanomyces root rot, bruchidsand lima bean pod borer. Lupin lines with horizontal resistanceto fusarium wilt are sought along with sources of high nitrogenfixing ability. Genetic research includes that on analysis ofgenetic diversity and determination of the genetic control ofimportant agronomic traits, including disease and stress resis-tances and genetic adaptation (Chekalin and Alpatiev 1988).The work of R. Makasheva on selection characters of peas basedon Makasheva (1973) was translated into English and otherslanguages. Breeding low-alkaloid, perennial lupin forms(Lupinus polyphyllus Lindl.) was also developed (Chekalin andKurlovich 1989).

Ecogeographical investigations initiated by Vavilov havemade it possible to create valuable materials through hybridiza-tion of forms with different characters under different condi-tions. One example is a method for obtaining transgressiveforms of lupin based on an ecogeographical approach (Kurlovichet al. 1995; Rep’ev and Barulin 1998). We have shown that eachquantitative character has from two to five or more types ofvariability (Rep’ev 1988). It was not always appreciated that, fora majority of accessions, plant characters change under differ-ent conditions. Hybrid progeny can behave similarly to theparental forms when crosses have involved parents with identi-cal types of variability for a character. However, hybrid progenyinclude transgressive forms when the result of crosses betweenparents with different types of variability. The distinctions invariability of characters in parental forms can be based ontesting under different conditions. Investigations of this phe-nomenon can lead to identification of valuable transgressiveforms for many characters, including chemical composition anddisease resistance (Kurlovich et al. 1995). Increased resistance oflupin to fusarium wilt is an example (Figs. 3 and 4). Lupincultivars and lines (547 accessions) were tested for fusariumresistance under different environmental conditions in two re-gions in Russia (near Bryansk and St. Petersburg) and in Ukraine(near Kiev) on plots with artificially infected soil. A large num-

Fig. 3. Variation in the degree of fusarium wilt susceptibilityof two cultivars of Lupinus angustifolius and their transgres-sive form on plots with artificially infected soil in differentregions of Russia and Ukraine.

Fig. 4. Variation in the degree of fusarium wilt susceptibilityof two lines of Lupinus luteus and their transgressive formon plots with artificially infected soil in different regions ofRussia and Ukraine.


ber of accessions of different lupin species were selected for theirresistance based on results from a single plot with infected soil.Yet on two other plots they were susceptible. Differences in thedisease susceptibility of the same accessions were thus found incontrasting environments. Resistant forms from one region werecrossed with accessions resistant in two other regions. Thisresulted in two transgressive resistant forms in the F4. Crosseswere ‘Frost’ x ‘Apendrilon’ (L. angustifolius) and line G-413 x line85 (L. luteus). Their resistance in all three regions appeared to behigher than that of their parental forms (Fig. 3 and 4). These twotransgressive forms with increased resistance to fusarium wiltwere found suitable for inclusion in the breeding program offusarium resistant forms in Russia, Belarus and Ukraine.

This interesting phenomenon was commented on by Ride(1992), in which the author summarized what was known aboutthe recognition and response mechanisms of higher plants tofungi. Study of the specific incompatibility between races ofpathogens and certain host cultivars has allowed rapid progressto be made in understanding the mechanism underlying success-ful infection (compatibility) and resistance (incompatibility).

Results from our experiments indicate why transgressiveforms occur in the breeding process only very seldom, acciden-tally, and among large numbers of hybrid materials. Our ap-proach to breeding makes the process of obtaining transgressivesmore controlled and effective. We have also tested Dragavtsev’sideas about limiting environmental factors on leguminous crops(Dragavtsev 1997) for transgressive selection. Using ourecogeographical approach, transgressive forms of vetch, peaand chickpea were also obtained (Rep’ev 1988). Vavilov’s geo-graphical and ecological approaches have been widely used anddeveloped by other scientists (Korsakov 1971; Chekalin andAlpatiev 1988; Tigerstedt 1994; Hill et al. 1998).

Vavilov foresaw the disappearance of many valuable formsof plants under the influence of human activity. Luckily, manyspecies and forms were saved following the expeditions ofVavilov and his colleagues. Many scientists from Brazil, Ethio-pia, Israel, Portugal and elsewhere have asked VIR for acces-sions of leguminous crops Vavilov collected in their countries.These genetic resources only existed in the collection of VIRthanks to Vavilov, and are now accessible for all to use. Thetraditions and methods of Vavilov are continued by numerousscientists in many countries. In the field of leguminous crops,the ideas of Vavilov have been further developed in Australia(Gladstones 1974, 1998; Sweetingham 1986, 1989; Cowling1994), Finland (Koskenmäki 1994; Hovinen 1994; Tigerstedt1994), Germany (Diederichsen and Hammer 1996), Italy(Laghetti et al. 1996; Saccardo 1996), Poland (Swìcicki 1988;Kazimierski and Kazimierska 1992, 1994), Portugal (Mota 1984;Tavares de Sousa et al. 1992; Neves Martins 1994), Sweden (Blixt1970, 1996), UK (Polhill 1976; Bisby 1981; Ambrose 1996) andmany other countries.

ConclusionsVavilov was the first to recognize the necessity for intensiveplant collecting and preservation. He was a highly qualifiedcollector of plant genetic resources and manager of collectingmissions, and an author of many theoretical and practical ideas

in the field of the global genetic diversity of cultivated plants. Amajor contribution of Vavilov was his ability to translate hisrapidly growing scientific knowledge in genetic resources intoeconomic use. Vavilov’s plant collecting expeditions served asthe basis for the leguminous crop collection in VIR, whichprovided the initial materials for over 75% of new grain legumecultivars created in Russia and other countries of the formerUSSR (Kurlovich 1996).

It is very difficult to do justice to Vavilov in this short article.The scope of Vavilov’s interests was extraordinarily wide andincluded practically all agricultural crops and disciplines ofplant science. The memory of Vavilov has been preservedthrough his collections of plant genetic resources, ideas, booksand followers. These are an important legacy of N.I. Vavilov,and were recently documented posthumously in his book ‘FiveContinents’ (Vavilov 1962, 1987a, 1997).

AcknowledgementsWe wish to express our gratitude to Prof. M.G. Agaev and DrsA.K. Stankevich and O.N. Korovina for the help, valuable coun-cil and participation in the development of intraspecific classifi-cations for leguminous crops.

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Impact of cultivation on active constituents of themedicinal plants Podophyllum hexandrum andAconitum heterophyllum in SikkimPankaj PrasadG. B. Pant Institute of Himalayan Environment and Development Sikkim Unit, P.O. Tadong, Sikkim- 737 102, India.Tel.: ++91-3592- 31673 (office); Fax: ++91-592-31090 (office); E-mail: [email protected]

SummaryImpact of cultivation on activeconstituents of the medicinalplants Podophyllum hexandrumand Aconitum heterophyllum inSikkimPodophyllum hexandrum Royle and Aconi-tum heterophyllum Wall. are herbaceousspecies that are important sources ofmedicines in India. These species arefound only in restricted pockets in theHimalayas. Habitat loss, combined withover-exploitation by pharmaceuticalcompanies, has led to the disappearanceof these species from many areas. Thereis thus a need for immediate conserva-tion. To assist in conservation these plantspecies have been cultivated ex situ in asubalpine garden at Kyongnosla Sikkim(2800 masl). The aim of this study was toassess the levels of podophyllotoxin andresin in Podophyllum and of aconitine andalkaloids in Aconitum in plants growingin situ and in the subalpine garden. Thesecompounds were abundant in wildplants. There were only minor statisti-cally significant differences in the con-tents of active components in cultivatedplants. The two species studied can beconsidered as a model for conservationof other important threatened species.

Key words: Aconitine, Aconitumheterophyllum, alkaloids, endangered,ex situ, in situ, podophyllotoxin,Podophyllum hexandrum, resin

ResumenEfectos del cultivo sobre losconstituyentes activos de lasplantas medicinalesPodophyllum hexandrum yAconitum heterophyllum enSikkimPodophyllum hexandrum Royle y Aconi-tum heterophyllum Wall. son especies her-báceas importantes como base de me-dicinas en la India. Estas especies se en-cuentran únicamente en algunos reduc-tos del Himalaya. La pérdida del hábitat,junto con la sobreexplotación por com-pañías farmacéuticas, ha hecho que estasespecies desaparezcan de muchas co-marcas. Es preciso pues tomar medidasinmediatas de conservación. Para contri-buir a su conservación, estas especiesvegetales se han cultivado ex situ en unjardín subalpino en Kyongnosla Sikkim(2800 m de altitud). El propósito de esteestudio era comprobar los niveles depodofilotoxina y resina en la Podophyllumy de aconitina y alcaloides en la Aconitumen plantas crecidas in situ y en el jardínsubalpino. Estos componentes eranabundantes en las plantas silvestres.Había sólo diferencias apreciables es-tadísticamente menores en el contenidode componentes activos en las plantascultivadas. Las dos especies estudiadaspueden considerarse como un modelopara la conservación de otras especiesimportantes amenazadas.

ARTICLE

RésuméEffet de la culture sur lescomposants actifs des plantesmédicinales Podophyllumhexandrum et Aconitumheterophyllum au SikkimPodophyllum hexandrum Royle et Aconi-tum heterophyllum Wall. sont des espècesherbacées médicinales importantes enInde. Ces espèces n’existent que dans deszones restreintes de l’Himalaya. La ré-duction de l’habitat combinée à la surex-ploitation par les compagnies pharma-ceutiques a conduit à la disparition de cesespèces dans de nombreux sites. Il y adonc un besoin urgent de préservation.Dans ce but, ces espèces ont été cultivéesex situ dans le jardin subalpin de Kyong-nosla Sikkim (2800 m d’altitude).L’objectif de cette étude a été de déter-miner les taux de podophyllotoxine et derésine dans Podophyllum et d’aconitine etd’alcaloïdes dans Aconitum pour des plan-tes poussant in situ et cultivées dans lejardin subalpin. Ces composants se sontrévélés abondants dans les plantes sau-vages. Des différences statistiquementsignificatives mineures ont été notéesavec les valeurs des composants actifsdes plantes cultivées. Ces deux espècespeuvent être considérées comme unmodèle à suivre pour la préservationd’autres espèces importantes menacées.

IntroductionSikkim covers an area of 7096 km2 in northeast India and is abiological ‘hot spot’ containing about 5000 species of floweringplants (Hajra and Verma 1996). J. D. Hooker (1983) made thefirst study of the flora of the Sikkim Himalayas during 1871-1897, and this was followed by a comprehensive work onmedicinal plants of Darjeeling and Sikkim by Biswas (1956).Over 400 plants possessing therapeutic properties have beenrecorded from the region (Srivastava and Kapaki 1990). Rai andSharma (1994) made studies on the status, uses and potentialof 40 important medicinal plants of the Sikkim Himalayas.Sikkim’s medicinal plants have been used in Ayurvedic andTibetan medicines, and their status and cultivation techniqueshave been carefully recorded (Nautiyal 1995; Sharma et al. 1995;Singh 1995; Trogawa et al. 1995; Tsarong 1995).

Indiscriminate collection of medicinal plants has led tomany of them becoming rare, threatened or endangered. Podo-

phyllum hexandrum Royle and Aconitum heterophyllum Wall are herba-ceous, rhizomatous species of great medicinal importance thatare now endangered in India (Nayar and Sastry 1990). Bothspecies are distributed in restricted pockets throughout the al-pine Himalayan region. The rhizome of P. hexandrum yields podo-phyllotoxin and resin, and that of A. heterophyllum aconitine andalkaloids. Continued extraction of rhizomes of these two speciesby pharmaceutical companies and practitioners of ethnic medi-cine has led to the virtual disappearance of the two species fromthe Himalayan region. Traditional herbal mixtures are frequentlyprepared through processes of drying, crushing, heating, boil-ing, and sometimes reducing to ash. The complex approach topharmacology is based on a concept of “synergistic activity” ofthe multiple components in a traditional formula. However,with increasing interest in traditional ethnopharmacy andherbal medicine and increasing preference for natural substances


in health care, the natural stocks of these valuable plants, andmany additional species, are under great pressure.

In an attempt at ex situ conservation and standardization ofcultivation techniques, the Forest Department of Sikkim hasgrown these two species in a subalpine garden at Kyanongsla(2800 masl). The investigations were carried out on plants fromthe subalpine garden and plants from the wild. The studyaimed at evaluating the impact of cultivation and low-altitudeacclimatization on levels of active constituents in their rhi-zomes. Little work has been done previously on estimation ofactive constituents of these species with respect to location of exsitu conserved populations.

Materials and methodsThe rhizomes of P. hexandrum and A. heterophyllum were collected fromwild plants growing in an alpine region and cultivated plants fromthe subalpine garden of Kyanongsla (n=10). Samples were dried ina hot-air oven at 40°C for 72 hours and were then powdered foranalyses. Resin was extracted from the dry powder of P. hexandrumrhizomes using ethanol in a Soxhlet apparatus. The ethanol extractwas distilled under vacuum to leave a residue that was dissolved inethanol and then precipitated with weak acid. The precipitate wasfiltered and weighed to obtain resin content. Podophyllotoxin wasdetermined by dissolving resin samples in HPLC-grade methanol.A Beckman System Gold HPLC with a silica packed column (C-18;4.5x250 mm; λmax 290) was used with methanol (1 ml/min) as themobile phase. Podophyllotoxin from Sigma Chemicals was used toestablish the calibration curve. Active constituents were extractedfrom the dry powder of A. heterophyllum rhizome using 90% ethanolin a Soxhlet apparatus. The extract was evaporated under vacuumto get a dark residue that was further extracted in hexane andevaporated. The residue was then suspended in chloroform andextracted several times using 2% sulphuric acid. The chloroformlayer gave a neutral fraction, while neutralisation of the acidicextract (sodium carbonate at pH 5) and extraction with chloroformgave a crude alkaloid fraction, which was dried and weighed.Aconitine was estimated using the crude alkaloid (0.1 mg) dis-solved in 10 ml of hexane, chloroform and methanol (70:20:10),and determined in a Beckman System Gold HPLC with a silicapacked column (C-18; 4.5 x 250 mm; λmax 240) and hexane/chloroform/methanol (1 ml/min) as the mobile phase. Aconitine

from Sigma Chemicals was used to establish a calibration curve.Data were analyzed statistically with ANOVA (Snedecor andCochran 1967).

Results and discussionActive constituents of P. hexandrum (resin and podophyllotoxin)and A. heterophyllum (alkaloids and aconitine) from both wild(alpine region) and cultivated (subalpine region) plants aregiven in Figs. 1 and 2. Active constituents of both the plantspecies were significantly lower in cultivated plants than in wildplants (P. hexandrum: resin F1, 18=336, P < 0.0001, podophyllotoxinF1, 18=1388, P < 0.0001; A. heterophyllum: alkaloids F1, 18=67, P <0.0001, aconitine F1, 18 =2731, P < 0.0001). However, differencesin actual amounts of each type of active constituent were small.These differences, which were consistent for all active constitu-ents, could have resulted from moving the plants from an alpineto a subalpine environment or from growing wild rather thanhaving been cultivated.

The results suggest that these two species can be cultivatedat moderate elevations, the accompanying decrease in activeconstituents in the cultivated forms being fairly small. There-fore, cultivation of these species under semi-natural conditions,as in the subalpine garden at Kyanongsla, can meet the de-mands of the pharmaceutical companies and ethnic medicine,while ex situ conservation in gardens and in situ conservation innature could be undertaken.

A policy of rotational harvesting from demarcated areasthrough permits would allow recuperation and sustainable har-vest. Most of the medicinal plants occur in restricted areas ofSikkim and collection permits are issued only to local inhabit-ants. This has restricted activities of collectors from outside thearea. The Forest Department has also been returning some ofthese medicinal plants to their natural habitat to gauge whethersustainable harvesting in demarcated areas can meet the re-quirements for traditional use. The remaining areas in the wildshould be set aside for in situ conservation and harvesting thereshould cease until they are adequately reestablished.

AcknowledgementThe author thanks the Director of the G.B. Pant Institute ofHimalayan Environment and Development. I am grateful to the

Fig. 1. Mean (±s.e.) resin and podophyllotoxin contents inthe rhizomes of Podophyllum hexandrum collected inSikkim from a cultivated subalpine region (shaded) and awild alpine region (unshaded).

Fig. 2. Mean (±s.e.) alkaloid and aconitine contents in therhizomes of Aconitum heterophyllum collected in Sikkimfrom a cultivated subalpine region (shaded) and a wildalpine region (unshaded).


Director of the High Altitude Plant Physiology Research Center,H.N.B.G.U Srinagar (Garhwal), UP for HPLC analysis, and alsoacknowledge the courtesy of Dr. Eklabya Sharma for reviewingthe manuscript. The Forest Department, Government of Sikkim,is thanked for permission to work in the Kyanongsla AlpineGarden.

ReferencesBiswas, K. 1956. Common Medicinal Plants of Darjeeling and

Sikkim Himalaya. Bengal Government Press, West Bengal.Hajra, P.K. and D.M. Verma. 1996. Flora of Sikkim. Botanical

Survey of India, Calcutta.Hooker, J.D. 1983. Flora of British India (1871–1897), Vols. I-VII.

Bishen Singh Mahendra Pal Singh, Dehra Dun, India.Nautiyal, M.C. 1995. Agro-technique of some high altitude me-

dicinal herbs. Pp. 53-64 in Cultivation of Medicinal Plantsand Orchids in Sikkim Himalaya (R.C. Sundriyal and E.Sharma, eds.). Bishen Singh Mahendra Pal Singh, Dehra Dun,India.

Nayar, M.P. and A.P.K Sastry. 1990. Red Data Book of IndianPlants. Botanical Survey of India, Calcutta.

Rai, L.K. and Sharma, E. 1994. Medicinal Plants of the SikkimHimalaya: Status, Usage and Potential. Bishen Sing MahendraPal Singh, Dehra Dun, India. 152 pp.

Sharma, E., L.K. Rai, S.T. Lachungpa and R.P. Awasthi. 1995.Status of medicinal plants and their cultivation potential inSikkim, Pp. 43-51 in Cultivation of Medicinal Plants andOrchids in Sikkim Himalaya (R.C. Sundriyal and E. Sharma,eds.). Bishen Singh Mahendra Pal Singh, Dehra Dun, India.

Singh, D.N. 1995. Use of medicinal plants of Sikkim in Ayurvedicmedicine. Pp. 65-68 in Cultivation of Medicinal Plants andOrchids in Sikkim Himalaya (R.C. Sundriyal and E. Sharma,eds.). Bishen Singh Mahendra Pal Singh, Dehra Dun, India.

Snedecor, G.W and W.G. Cochran. 1967. Statistical Methods.Oxford & IBH, New Delhi.

Srivastava, T.N. and B.K. Kapaki. 1990. Resource survey ofplants of potential economic value of Sikkim Himalaya. Bull.Medico-Ethno-Botany Res. 12(1–2):1–11.

Trogawa, T.G., J. van der Waals and N. de Jong. 1995. Conditionsfor replanting and conserving high valued Tibetan medicinalherbs. Pp. 69-73 in Cultivation of Medicinal Plants and Or-chids in Sikkim Himalaya (R.C. Sundriyal and E. Sharma,eds.). Bishen Singh Mahendra Pal Singh, Dehra Dun, India.

Tsarong, T.J. 1995. Tibetan medicinal plants: an agenda forcultivation. Pp. 75-79 in Cultivation of Medicinal Plants andOrchids in Sikkim Himalaya (R.C. Sundriyal and E. Sharma,eds.). Bishen Singh Mahendra Pal Singh, Dehra Dun, India.

36 Plant Genetic Resources Newsletter, 2000, No. 124 Plant Genetic Resources Newsletter, 2000, No. 124: 36 - 40

Morpho-agronomic variability of the diploid wheatTriticum monococcum L.S. Empilli, R. Castagna and A. Brandolini*Istituto Sperimentale per la Cerealicoltura, Via Mulino 3, 26866 S. Angelo Lodigiano, Italy. Email: [email protected]

SummaryMorpho-agronomic variabilityof the diploid wheat Triticummonococcum L.The A-genome diploid wheat, Triticummonococcum, is among the first plants cul-tivated by humans. In spite of severalfavourable characteristics (high proteincontent, resistance to diseases, adaptabil-ity to difficult environments), its useful-ness is very limited because of poor ag-ronomic performance and non free-threshing seeds. The S. Angelo Lodigianosection of the Istituto Sperimentale per laCerealicoltura, Italy, in co-operation withthe Max-Planck Institute, Cologne, Ger-many, is currently evaluating the geneticvariation of this diploid wheat to identifyaccessions with useful traits. In this study,1039 entries of Triticum monococcumsubsp. monococcum, subsp. boeoticum andsubsp. sinskajae were characterized for 17morphological and agronomic descrip-tors. A broad variation for all the traitsstudied was detected and promising ac-cessions for breeding purposes wereidentified.

Key words: Boeoticum, einkorn,genetic variability, monococcum,sinskajae, Triticum

ResumenVariabilidad morfo-fisiólogicay cualitativa del trigo diploideTriticum monococcumEl trigo diploide de genoma A Triticummonococcum es una de las primeras plan-tas cultivadas por la humanidad. Aunquetenga caracteristicas favorables (alto con-tenido proteico, resistencia a enfer-medades, adaptación a ambientes difer-entes), su importancia es muy limitadapor sus pobres prestaciones agronómi-cas y su semilla envuelta en las glumas.La sección de S. Angelo Lodigiano delIstituto Sperimentale per la Cerealicoltu-ra, en cooperación con el Max-Planck In-stitut de Colonia, està evaluando la vari-ación genética de este trigo diploide paraidentificar muestras con caracteristicasútiles. En este estudio, 1039 muestras deTriticum monococcum subsp. monococcum,subsp. boeoticum y subsp. sinskajae fueroncaracterizadas mediante 17 descriptoresmorfo-fisiológicos, agronómicos y cuali-tativos. Todas las caracteristicas estudia-das presentaron una alta variabilidad;además, se encontraron muestras con ungran potencial para programas de mejo-ra genética de trigo.

RésuméVariabilité morpho-agronomiquechez le blé diploïde Triticummonococcum L.Le blé diploïde de génome A, Triticummonococcum L., fait partie des premièresplantes cultivées par les hommes. Endépit de plusieurs caractéristiques favor-ables (teneur protéique élevée, résistanceaux maladies, adaptation aux environne-ments difficiles), son utilité reste très lim-itée par la faiblesse de ces performancesagronomiques et par la non-libérationde ses graines au battage. La section S.Angelo Lodigiano de l’Institut expéri-mental de céréaliculture, en Italie, en col-laboration avec l’Institut Max-Planck deCologne en Allemagne, poursuitl’évaluation de la variation génétique dece blé diploïde afin d’identifier des acces-sions aux caractères utiles. Dans cetteétude, 1039 entrées de Triticum monococ-cum subsp. monococcum, subsp. boeoticumet subsp. sinskajae ont été caractériséespour 17 descripteurs morphologiques etagronomiques. Une importante varia-tion a été détectée pour tous les car-actères étudiés et des accessions prom-etteuses en termes de sélection ont étéidentifiées.

IntroductionEinkorn (Triticum monococcum L.) is a diploid A-genome wheat(2n=2x=14) from the Near East, domesticated about 10 000years ago in southwest Turkey in the Karaca Dag mountains(Heun et al. 1997). Widely harvested (wild form) and cultivated(domesticated form) by Neolithic farmers in the Fertile Crescentregion and afterwards in Europe, its importance decreased afterthe Bronze age (Zohary and Hopf 1993), when higher yieldingand free-threshing tetraploid and hexaploid wheats replaced it.Today einkorn is cultivated only in small areas of the Mediterra-nean region (Perrino et al. 1996), while its wild form was stillcommon in some locations in Turkey during the 1960s (Zoharyand Hopf 1993). MacKey (1988) identifies two Triticummonococcum subspecies: monococcum (domesticated) and boeoticum(wild). Filatenko and Kurkiev (1975) described also a domesti-cated, free-threshing form, subsp. sinskajae .

A-genome diploid wheats are important for their role in thephylogenesis of the polyploid wheats and for the relative sim-plicity of their genome, features that make them ideal for ge-netic studies. The diploid wheats are also used in bread anddurum wheat breeding programmes, because of their resistanceto several biotic and abiotic stresses (Gerechter-Amitaj et al. 1971;Potgieter et al. 1991; Dyck and Bartoš 1994). Therefore, the evalu-ation, identification and description of the genetic variation of

einkorn accessions stored in germplasm collections is of utmostimportance for the prevention of genetic erosion and to promoteits use in breeding programmes.

Several studies on limited numbers of accessions have re-vealed high variability for morphophysiological traits (Sharma etal. 1981; Waines 1983), yield (Guzy et al. 1989; Vallega 1992;Castagna et al. 1995), pathogen resistance (Gill et al. 1983), storageproteins (Waines and Payne 1987; Saponaro et al. 1995), starchcharacteristics (Taira et al. 1995; Fujita et al. 1996), bread-makingquality (Borghi et al. 1996) and molecular markers (Vierling andNguyen 1992; Takumi et al. 1993; Castagna et al. 1994; Heun et al.1997).

The S. Angelo Lodigiano section of the Istituto Sperimentaleper la Cerealicoltura, Italy, in collaboration with the Max-PlanckInstitute, Cologne, Germany, assembled 1039 einkorn acces-sions from several world gene banks. The objectives were:l to evaluate the genetic variation among accessions and col-lect preliminary information for the constitution of a core collec-tion;l to verify the correspondence between taxonomic classifica-tion and variability observed; andl to identify accessions useful for an einkorn breedingprogramme.

ARTICLE


Materials and methodsThe 1039 einkorn accessions studied, divided bytaxonomic classification and origin, are presented inTable 1. One hundred seeds of each accession werehand planted in the field at S. Angelo Lodigiano,Italy (lat 45°N) in autumn (6 November 1992) and inspring (15 March 1993) in single rows 1.50 m longand 0.50 cm apart. The spring planting was neededto evaluate seasonality. Nitrogen was applied beforeplanting (60 kg/ha) and at tillering (30 kg/ha).Plants were harvested by hand between 15 and 30July 1993. The einkorns were characterized using 16morpho-agronomic descriptors (Tables 2 and 3), in-cluding some suggested by IBPGR (1985). In addi-tion, the SDS sedimentation test was performed fol-lowing Preston et al. (1982) to assess bread-makingquality. Statistical analysis was carried out using thestatistical package MSTAT-C (1991).

Results and discussionMean, minimum and maximum values and stan-dard deviations of the eight continuously variabledescriptors showing are shown in Table 2, while theirfrequency distributions are depicted in Fig. 1. Thevariability of the descriptors showing discontinuousvariation is presented in Table 3.

Most of the einkorns had a facultative growthhabit, flowering equally well when sown in autumnand in spring. All the winter growth habit accessionswere from the wild subspecies boeoticum. Heading

Table 3. Frequency distribution (%) for the discontinuously variable descriptors for Triticum monococcum

Descriptor Class 0 1 2 3 4 5 7 9

Seasonality 1 = winter; - 4.6 95.4 - - - - -2 = facultative

Number of nodes 1 = 3 nodes; - 66.4 31.0 2.6 - - - -2 = 4 nodes;3 = 5 nodes

Stem section 1 = empty; - 93.5 6.5 0.0 - - -2 = semi-full;3 = full

Leaf hairiness 1 = absent; - 34.9 65.1 - - - - -2 = present

Awns 0 = none; 3 = short; 0.0 - - 0.6 - - 99.4 -7 = long

Spike density 1 = very lax; 3 = lax; - 0.0 - 0.1 - 64.3 35.2 0.45 = intermediate;7 = dense;9 = very dense

Seed colour 1 = white; 2 = red; - 0.0 57.4 0.5 34.7 7.4 - -3 = purple;4 = red-green;5 = green-black

Lodging 0 = no lodging; 35.2 - - 30.6 - 15.9 18.3 -susceptibility 3 = little lodging;

5 = lodging;7 = very lodging

Powdery mildew from 0 = none to 98.5 0.5 0.1 0.4 0.2 0.2 0.1susceptibility 7 = high

Table 1. Origin of the Triticum monococcum accessionscharacterized at s. Angelo Lodigiano, Italy

Origin subsp. subsp. subsp. Totalmonococcum boeoticum sinskajae

Unknown 93 21 1 115Balkans 95 12 107Caucasus 5 6 2 13Europe† 100 16 - 116Iran - 33 - 33Iraq - 344 - 344Lebanon-Israel 2 8 - 10Morocco 13 - - 13Turkey 29 259 - 288Total 337 699 3 1039

†Austria, France, Germany, Italy, Spain, Sweden, Switzerland and UK

Table 2. Mean, maximum, minimum and values and standarddeviations for eight continuously variable descriptors forTriticum monococcum

Descriptor Mean Minimum Maximum Standarddeviation

Heading date 28.6 7 46 6.30(days from 1 May)Plant height (cm) 101.6 45 135 12.92Peduncle length (cm) 48.7 18 70 7.97Spikelets/spike 25.3 12 44 8.13Seed length (mm) 7.9 6 10 0.81Seed width (mm) 2.6 2 4 0.481000-seed weight (g) 23.7 11 53 5.32SDS sedimentation 40.0 12 98 18.83test (ml)


date was very variable, with a difference of 39 days between theearliest and the latest accessions. The subsp. boeoticum samplesheaded on average 10 days before cultivated einkorns; however,a few early subsp. monococcum accessions had heading datessimilar to those of subsp. boeoticum and of bread wheat. Earlymaterials are of utmost importance in breeding programmesaimed at the Mediterranean region; the long life cycle of einkorn,extending well into the dry, hot summer, severely limits poten-tial yields (Castagna et al. 1996).

Plant height was very variable. Subspecies boeoticum had thetallest plants (135 cm), while subsp. sinskajae and subsp.monococcum had the shortest plants (65-85 cm). The shortestaccession (45 cm) came from a mutation breeding programme.Peduncle length varied widely and was correlated to plantheight. Node number ranged between three and five, with mostof subsp. boeoticum accessions having few nodes and long intern-odes. Short accessions are of particular interest for breedingprogrammes because tall, thin stems increase lodging near har-vesting time. Despite their height, many accessions (65.8%)showed little or no lodging; however, this was recorded on singlerows and during one year only and may thus not be an accurate

reflection of likely performance in thefield.

Leaf hairiness, a taxonomic classifi-cation trait, is evident in subsp.boeoticum accessions, but is normally ab-sent in subsp. monococcum and subsp.sinsksjae . However, a few exceptions werefound, possibly because ofmisclassification or of the existence ofintermediate types between subspecies.

Spike density neatly differentiatedthe cultivated einkorns from their wildrelatives: subsp. boeoticum had lax tomedium-lax spikes, while subsp.monococcum had dense spikes and subsp.sinsksjae presented very dense ones.Number of spikelets per spike was veryvariable and revealed a marked bimodaldistribution (Fig. 1), with a mean valuehigher for subspp. monococcum andsinskajae (35 spikelets/spike) than forsubsp. boeoticum (20 spikelets/spike).The awns were well developed in allaccessions except in the three sinskajaesamples.

Seed length and width were moder-ately variable, with subspp. monococcumand sinskajae having shorter and broaderseeds than subsp. boeoticum. Average1000-seed weight was 23.7 g, muchlower than that of bread or durumwheats (commonly 40-45 g/1000seeds); the 13 accessions with a 1000-seed weight above 40 g where all fromthe subsp. monococcum group. The mostfrequent seed colour was red, although

subsp. boeoticum showed also very deep red-green and green-black seeds.

The vast majority of einkorns tested did not show suscepti-bility to powdery mildew, confirming the widespread pathogenresistance reported in literature (Vallega 1992; Bai et al. 1998;Udachin 1998); the few susceptible accessions were all from thesubsp. boeoticum group. Most accessions showed low SDS sedi-mentation, indicating poor bread-making quality, but valuesranged from 12 to 98 ml among accessions. Forty-eight acces-sions of wild einkorn and eight accessions of cultivated einkornhad SDS greater than 80 ml, comparable to that of the high-quality bread wheat Salmone grown under the same experimen-tal conditions. The possibility of using einkorn flour in breadmaking has been recently confirmed by Corbellini et al. (1999).

The correlations among some of the traits are reported inTable 4. Most of the correlations are significant because of thelarge number of observations, hence, only values above 0.50 willbe discussed here.

Heading date was positively correlated with number ofspikelets per spike (r=0.71) and spike density (r=0.62), andnegatively correlated with peduncle length (r=-0.56). These cor-

Fig. 1. Frequency distribution of eight continuously variable descriptors for Triticummonococcum.


relations have a physiological explanation: delayed headingallows the differentiation of a bigger sink organ as well asdevelopment of more and shorter internodes. Since the rachis isa transformed stem, delayed heading reduces the distance be-tween spikelets and increases spike density.

The negative correlation between heading date and leafhairiness (r=-0.64) is explained by taxonomy: early accessionsare mostly from subsp. boeoticum, characterized by high leafhairiness.

As expected, plant height was correlated with peduncle length(r=0.63), which is positively correlated with leaf hairiness (r=0.63;wild einkorns are hairy and with long peduncle) and negativelycorrelated with spike density (r=-0.59). The increase in stem inter-node length is paralleled by an increase in rachis internode length,leading to a laxer spike. Leaf hairiness is negatively correlatedwith spike density (r=-0.89), number of spikelets per spike (r=-0.81) and 1000-seed weight (-0.56), and positively correlated withseed colour (r=0.61). Similarly, spike density is positively corre-lated with number of spikelets per spike (r=0.80) and with 1000-seed weight (r=0.58), and negatively correlated with seed colour(r=-0.58). All these correlations have a taxonomic basis sincesubsp. monococcum accessions differ from subsp. boeoticum, havingdenser spikes with a higher number of spikelets, heavier andlighter-coloured seeds, and no leaf hairs.

ConclusionsIdentification and description of the genetic variability availablein germplasm collections are the basis of improved plans de-signed to control genetic erosion; they are also a preliminaryrequirement for the exploitation of useful traits in plant breed-ing. Strongly adhering glumes, excessive height, late headingand small seeds are the prevalent characters limiting einkorncultivation in the Mediterranean environments. However, thediploid wheats characterized in this study showed a broadvariation for these and other traits, which allows for the identi-fication of promising accessions for einkorn breeding.

In a few cases the recorded passport classification did notagree with the data collected, suggesting the need for the re-grouping of some accessions. A few samples (ID183, ID223,ID356 and ID557) showed taxonomic characters intermediatebetween wild and cultivated einkorns.

AcknowledgementsWe acknowledge with gratitude the following genebanks anddonors:Max Planck Institut, Cologne, Germany; Institut für Genetik undKulturpflanzen Forschung, Gatersleben, Germany; Institut fürPflanzenbau und Pflanzenzüchtung, Braunschweig, Germany;Istituto di Genetica e Sperimentazione Agraria N. Strampelli,Lonigo, Italy; Istituto del Germoplasma, Bari, Italy; IstitutoNacional de Tecnologia Agropecuaria, Argentina; Vavilov AllUnion Institute of Plant Industry, Saint Petersburg, Russia; Insti-tute de recherches agronomiques, Nyon, Switzerland; CambridgeLaboratory, Norwich, England; Australian Winter Cereals Collec-tion, Tamworth, Australia; University of Alberta, Edmonton,Canada; Kansas State University, Manhattan, Kansas, USA;National Small Grains Collection, Aberdeen, Idaho, USA.Ta

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ReferencesAACC. 1995. Approved methods of the AACC. AACC, St Paul,

Minnesota, USA.Bai, D., D.R. Knott and J.M. Zale. 1998. The inheritance of leaf

and stem rust resistance in Triticum monococcum L. Can. J.Plant Sci. 78:223-226.

Borghi, B., R. Castagna, M. Corbellini, M. Heun and F. Salamini.1996. Breadmaking quality of einkorn wheat (Triticummonococcum subsp. monococcum). Cereal Chem. 73:208-214.

Castagna, R., B. Borghi, M. Heun and F. Salamini. 1996. Inte-grated approach to einkorn breeding. In Hulled wheats. Pro-ceedings of the First International Workshop on HulledWheats, 21-22 July 1995, Castelvecchio Pascoli, Tuscany,Italy (S. Padulosi, K. Hammer and J. Heller, eds.). Promotingthe conservation and use of underutilized and neglected crops4. International Plant Genetic Resources Institute, Rome, Italy.

Castagna, R., B. Borghi, N. Di Fonzo, M. Heun and F. Salamini.1995. Yield and related traits of einkorn (T. monococcum subsp.monococcum) in different environments. Eur. J. Agron. 4:371-378.

Castagna, R., G. Maga, M. Perenzin, M. Heun and F. Salamini.1994. RFLP-based genetic relationships of einkorn wheats.Theor. Appl. Genet. 88: 818-823.

Corbellini, M., S. Empilli, P. Vaccino, A. Brandolini, B. Borghi, M.Heun and F. Salamini. 1999. Einkorn characterisation forbread and cookie production in relation to protein subunitcomposition. Cereal Chem. 76:727-733.

Dyck, P.L. and P. Bartoš. 1994. Attempted transfer of leaf rustresistance from Triticum monococcum and durum wheat tohexaploid wheat. Can. J. Plant Sci. 74:733-736.

Filatenko, A.A. and U.K. Kurkiev. 1975. A new species Triticumsinskajae A. Genetike i Selektsii. 54:239-241.

Fujita, N., A. Wadano, S. Kozaki, K. Takaoka, S. Okabe and T.Taira. 1996. Comparison of the primary structure of waxyproteins (granule-bound starch synthase) between polyploidwheats and related diploid species. Biochem. Genet. 35: 403-411.

Gerechter-Amitaj, Z.K., I. Wahl, A. Vardi and D. Zohary. 1971.Transfer of stem rust seedling resistance from wild diploideinkorn to tetraploid durum wheat by means of a triploidhybrid bridge. Euphytica 20:281-285.

Gill, B.S., L.E. Browder, J.H. Hatchett, T.L. Harvey, T.J. Martin,W.J. Raupp, H.C. Sharma and J.G. Waines. 1983. Diseaseand insect resistance in wild wheats. Pp. 785-791 in Proceed-ings of the Sixth International Wheat Genetics Symposium (S.Sakamato, ed.). Plant Germ-Plasm Institute, Kyoto Univer-sity, Japan.

Guzy, M.R., B. Hedaie and J. G. Waines. 1989. Yield and itscomponents in diploids, tetraploid and hexaploid wheats indiverse environments. Ann. Bot. 64:635-642.

Heun, M., R. Schaefer-Pregl, D. Klawan, R. Castagna, M. Accerbi,B. Borghi and F. Salamini. 1997. Site of einkorn wheat domes-tication identified by DNA fingerprinting. Science 278:1312-1314.

IBPGR. 1985. Descriptors for wheat (revised). IBPGR, Rome,Italy. 12 pp.

MacKey, J. 1988. A plant breeder’s perspective on taxonomy ofcultivated plants. Biol. Zentralbl. 107:369-379.

Mstat-C. 1991. Michigan State University, East Lansing, USA.Perrino, P., G. Laghetti, L.F. D’Antuono, M. Ajlouni, M.

Camberty, A.T. Szabo and K. Hammer. 1996.Ecogeographical distribution of hulled wheat species. InHulled wheats. Proceedings of the First International Work-shop on Hulled Wheats, 21-22 July 1995, CastelvecchioPascoli, Tuscany, Italy (S. Padulosi, K. Hammer and J. Heller,eds.). Promoting the conservation and use of underutilizedand neglected crops 4. International Plant Genetic ResourcesInstitute, Rome, Italy.

Potgieter, G.F., G.F Marais and F. du Toit. 1991. The transfer ofresistance to the Russian wheat aphid from Triticummonococcum L. to common wheat. Plant Breeding 106:284-292.

Preston, K.R., P.R. March and K.H. Tipples. 1982. Assessment ofthe SDS sedimentation test for the prediction of Canadianbread wheat quality. Can. J. Plant Sci. 62:545-553.

Saponaro, C, N. Pogna, R. Castagna, M. Pasquini, P. Cacciatoriand R. Redaelli. 1995. Allelic variation at the GliA1, GliA2and GluA1 loci and quality in diploid wheat Triticummonococcum evaluated with SDS sedimentation test. Genet.Res. 66:127-137.

Sharma, H.C., J.G. Waines and K.W. Foster. 1981. Variability inprimitive and wild wheats for useful genetic characters. CropSci. 21:555-559.

Taira, T., N. Fujita, K. Takaoka, M. Uematsu, A. Wadano, S.Kozaki and S. Okabe. 1995. Variation in the primary struc-ture of waxy proteins (granule-bound starch synthase) indiploid cereals. Biochem. Genet. 33:269-281.

Takumi, S., S. Nasuda, Y.G. Liu and K. Tsunewaki. 1993. Wheatphylogeny determined by RFLP analysis of nuclear DNA. 1.Einkorn wheat. Jap. J. Genet. 68:73-79.

Udachin, R. 1998. Interspecific differentiation of Triticum specieswith reference to resistance to leaf and yellow rust. Rachis17:45-49.

Vallega, V. 1992. Agronomical performance and breeding valueof selected strains of diploid wheat Triticum monococcum .Euphytica 61:13-23.

Vierling, R.A. and H.T. Nguyen. 1992. Use of RAPD markers todetermine the genetic diversity of diploid wheat genotypes.Theor. Appl. Genet. 84:835-838.

Waines, J.G. 1983. Genetic resources in diploid wheats: the casefor diploid commercial wheats. Pp. 115-122 in Proceedings ofthe Sixth International Wheat Genetics Symposium (S.Sakamato, ed.). Plant Germ-Plasm Institute, Kyoto Univer-sity, Japan.

Waines, J.G. and P.I. Paine. 1987. Electrophoretic analysis of thehigh molecular weight subunits of Triticum monococcum , T.urartu and the A-genome of bread wheat (T. aestivum ). Theor.Appl. Genet. 74:71.76.

Zohary, D. and M. Hopf. 1993. Domestication of plants in theOld World. The origin and spread of cultivated plants inWest Asia, Europe and the Nile Valley. Second edition.Clarendon Press, Oxford, UK.

Plant Genetic Resources Newsletter, 2000, No. 124 41Plant Genetic Resources Newsletter, 2000, No. 124: 41- 43

Polyphenol oxidase activity as an index forscreening mango (Mangifera indica L.) germplasmagainst malformationR.R. Sharma1*, C.N. Singh2, O.P. Chhonkar2, A.M. Goswami1 andS.K. Singh1

1Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute, New Delhi-110012, India.Tel. :+91- 011-5785214; Fax: +91-011-5766420; email: [email protected]. of Horticulture, CCS College, Muzaffar Nagar, UP, India

SummaryPolyphenol oxidase activity as anindex for screening mango(Mangifera indica L.) germplasmagainst malformationCatecholase and cresolase (PPO) enzymeactivity and phenolic content were mea-sured in 24 mango varieties in newlyemerged vegetative growth during theSeptember-October flush in 1997 and1998. Incidence of malformation was re-corded in March-April 1998 and 1999.Enzyme activity and phenolic contentvaried widely among varieties, and washighest in ‘Bhadauran’ and lowest in‘Tommy Atkins’. Floral malformationincidence was highest (53.75%) in‘Tommy Atkins’ and lowest in‘Bhadauran’ (1.10%). A strong positivecorrelation was found between the inci-dence of floral malformation and bothenzyme activity and phenolic content.Thus PPO activity can be used as a bio-chemical index for screening mangogermplasm against malformation.

Key words: Catecholase, cresolase,ex situ, floral malformation,germplasm screening, Mangifera in-dica L., mango, polyphenol oxidase

ResumenActividad de oxidasa depolifenol (PPO) como índicepara seleccionar germoplasmade mango (Mangifera indica L.)contra malformacionesSe midieron la actividad enzimática decatecolasa y cresolasa (PPO) y el con-tenido fenólico en 24 variedades demango en crecimiento vegetativo re-ciente durante el período de empuje deseptiembre-octubre en 1997 y 1998. Lafrecuencia de malformación se registróen marzo-abril de 1998 y 1999. La activ-idad enzimática y el contenido fenólicofueron muy variables según las var-iedades, desde un máximo en ‘Bhadau-ran’ hasta un mínimo en ‘Tommy At-kins’. La frecuencia de malformación flo-ral fue más alta (53,75%) en ‘TommyAtkins’ y más baja en ‘Bhadauran’(1,10%). Se encontró una fuerte correl-ación positiva entre la frecuencia de mal-formación floral por un lado y la activ-idad enzimática y el contenido fenólicopor otro lado. La actividad de PPOpuede usarse así como índice bioquími-co para seleccionar germoplasma demango contra malformaciones.

ARTICLE

RésuméActivité polyphénol oxydase entant qu’indice pour le criblagedu germoplasme de mangue(Mangifera indica L.) contre lesmalformationsLes activités enzymatiques de la catécho-lase et de la crésolase (PPO) et la teneuren phénols ont été mesurées sur 24variétés de manguiers en phase depoussée végétative durant le rougisse-ment de septembre-octobre en 1997 et1998. L’incidence des malformations a étéenregistrée en mars-avril 1998 et 1999.L’activité enzymatique et la teneurphénolique ont varié grandement suiva-nt les variétés avec des valeurs maxi-males pour ‘Bhadauran’ et minimalespour ‘Tommy Atkins’. L’incidence demalformation florale a été la plus élevéechez ‘Tommy Atkins’ (53.75%) et la plusfaible chez ‘Baudharan’ (1.10%). Uneforte corrélation positive a été déduiteentre l’incidence de malformation floraleet l’activité enzymatique et la teneurphénolique. On peut donc utiliserl’activité PPO comme un indice bio-chimique pour le criblage contre la mal-formation du germoplasme de mangue.

IntroductionIndia is considered the original home of mango (Mangifera indicaL.). There are about 1500 varieties of mango in the world, ofwhich about 1200 are found in India (Pandey 1998). However,only a dozen varieties are grown commercially around theworld. The Indian Agricultural Research Institute (IARI), NewDelhi, maintains 94 mango accessions collected from differentparts of the world. These are used breeding, propagation andphysiological studies.

Floral malformation is a major problem in mango, renderingmango cultivation unproductive in northern India and otherregions of the world (Pandey et al. 1977; Ram1991). Incidence ofmalformation is high in many exotic accessions and someindigenous accessions under northern Indian conditions (per-sonal observation). Mango breeders in northern India havedirected their research efforts to developing hybrids resistant tomalformation. Unfortunately, there is no currently effectivemethod for screening germplasm at the juvenile stage for resis-tance to floral malformation, a serious drawback given the 7–8-year juvenile phase of mango.

Phenolic compounds are believed to impart resistance to dis-eases in plants and polyphenol oxidase (catecholase andcresolase) enzyme has been reported to be responsible for in vivosynthesis and accumulation of these compounds (Mayer andHarel1979; Vaughan and Duke1984). Thus, the level of polyphe-nol oxidase (PPO) in the early years of plant growth or in the flushof vegetative growth may provide an estimate of synthesis ofphenolic compounds in the plants, which may in turn be corre-lated to susceptibility or resistance to floral malformation (Sharmaet al. 1994). However, this has not been studied in mango.

These investigations were conducted on newly emergedgrowth flush of mango cultivars to determine whether PPOlevels may be used to screen germplasm for resistance to floralmalformation at the nursery or pre-flowering stage.

Material and methodsBearing plants (12-15 years old) of 24 diverse varieties (bothindigenous and exotic) namely ‘Eldon, ‘Sensation’, ‘TommyAtkins’, ‘Langra’, ‘Chausa’, ’Dashehari’, ‘Amrapali’, ‘Mallika’,


‘Bhadauran’, ‘Extrema’, ‘Edward’, ‘Zill’, ‘Alphonso’, ‘Neelum’,‘Kurukkan’, ‘Fazli’, ‘Zardalu’, ‘Himsagar’, ‘Totapuri Red Small’,‘Rataul’, ‘Lal Sundri’, H-13-1, H-8-1 and ‘Ratna’, were selectedfor the study. Shoot tips (10-15 mm long) from the September-October flush in 1997 and 1998 were excised and polyphenoloxidase (both catecholase and cresolase) activity and total phe-nol content were determined. The number of healthy and mal-formed panicles on each variety were counted in March 1998and March 1999 and averaged to give the incidence of malfor-mation. Data for the two years were pooled and analyzed usinga randomized design. Correlations between PPO activity andphenolic compound content and between PPO activity andmalformation incidence were calculated.

Estimation of phenolic contentPhenolic content was measured using the Folin Ciocalteu re-agent method suggested by Slinkard and Singleton (1977) withslight modifications (Sharma et al. 1994) and represented as mg/g of fresh tissue weight.

Preparation of enzyme extractThe crude enzyme extract was prepared at 4oC according to theprocedure of Valero et al. (1989) with some modifications. Shoottips excised from newly emerged vegetative growth were choppedand mixed before preparing enzyme extract. One gram of choppedmaterial, together with 5 ml of 100 mM phosphate buffer (pH 7.3)containing 10 mM sodium ascorbate, was homogenized in ablender for 15 s, filtered through four layers of gauze and centri-

fuged at 3000 g for 30 min. The precipitate was re-extracted for 15min with 5 ml of 1.5% Triton-X-100, prepared in 100 mM phos-phate buffer (pH 7.3). The final volume of the extract was madeup to 25 ml with phosphate buffer (pH 7.3) and then the filtratewas centrifuged at 15 000 g for 1 hour. An ammonium sulphatefractionation was carried out and the fraction precipitating be-tween 45 and 95% saturation was collected and re-dissolved.Following dialysis, this solution was used as an enzyme source.

Enzyme assayBoth catecholase and cresolase activities were measured spectro-photometrically at 400 nm according to the procedure describedby Sanchez-Ferrer et al. (1988), with slight modifications.Catecholase activity was measured using 30 mM 4-methyl cat-echol (4MC) as substrate, made up in 10 mM sodium acetatebuffer (pH 4.5). Three ml 100 mM phosphate buffer (pH 7.3) wasadded to 1 ml crude enzyme extract and 1 ml substrate wasadded at zero time. The change in absorbance at 400 nm wasrecorded in a CL-1200 spectrophotometer. Cresolase activity wasmeasured in the same way, except that 4-methyl phenol (p-cresol)was used as substrate, made up in 10 mM phosphate buffer (pH7.0). Enzyme activity was represented as change in absorbance at400 nm/g of tissue weight per minute (∆ A400 g-1 min-1).

Results and discussionCatecholase and cresolase activity and phenolic content variedwidely among genotypes (Table 1). Catecholase activity washighest in ‘Bhadauran’ (1.868), closely followed by H-8-1

Table 1. Polyphenol oxidase activity, phenolic content and malformation incidence in different mango cultivars

Cultivar Polyphenol oxidase activity Phenolic content Malformation(∆ A400 g

-1 min-1) (mg/g fresh tissue incidence (%)weight)

Catecholase Cresolase

Bhadauran 1.863 1.136 28.22 1.10 (6.02)H-8-1 (Amrapali x Lal Sundri 1.859 1.113 27.80 2.96 (9.98)Dashehari 1.142 0.670 21.62 31.50 (34.14)Langra 1.126 0.665 21.59 31.62 (34.20)Kurukkan 1.116 0.656 21.69 31.22 (33.96)Fazli 1.113 0.650 21.02 32.60 (34.82)Sensation 0.998 0.521 17.80 37.88 (38.00)Mallika 0.991 0.502 17.29 38.72 (38.47)Aphonso 0.990 0.513 17.26 37.68 (37.88)Eldon 0.985 0.501 17.19 38.93 (38.59)Rataul 0.985 0.516 16.92 37.46 (37.76)Lal Sundri 0.936 0.440 15.39 42.51 (40.69)H-13-1 (Amrapali x Sensation 0.929 0.432 15.35 42.66 (40.74)Extrema 0.928 0.433 15.89 42.12 (40.16)Neelum 0.928 0.430 16.00 42.30 (40.57)Zill 0.923 0.429 15.29 42.92 (40.92)Edward 0.920 0.426 15.21 43.70 (41.38)Amrapali 0.917 0.420 15.21 43.77 (41.50)Totapuri Red Small 0.916 0.422 15.49 42.80 (40.86)Himsagar 0.902 0.419 15.44 42.00 (40.40)Zardalu 0.859 0.386 12.63 52.16 (46.26)Ratna 0.859 0.389 12.33 52.42 (46.38)Chausa 0.853 0.380 12.19 52.83 (46.61)Tommy Atkins 0.823 0.366 11.56 53.75 (47.18)P <0.05) 0.038 0.027 0.89 2.39

*Figures in parentheses are transformed values.


(1.849), and lowest in ‘Tommy Atkins’ (0.823), closely followedby ‘Chausa’ (0.853), ‘Zardalu’ (0.859) and ‘Ratna’ (0.859).Cresolase activity showed a trend similar to that for catecholaseexcept at lower absolute levels of activity (Table 1). This lowerobserved cresolase activity may have been due to a longer lagperiod in its production, its greater instability and rapid loss ofcresolase during extraction, as reported in grape by Wissemanand Lee (1980), Nakamura et al. (1983) and Sharma et al. (1994).Phenolic content followed a pattern similar to that forcatecholase and cresolase activity (Table 1). Shoot tips of‘Bhadauran’ contained the most phenols (28.22 mg/g freshtissue weight), followed by H-8-1 (27.80 mg/g), while ‘TommyAtkins’ contained the least phenols (11.56 mg/g), closely fol-lowed by ‘Chausa’ (12.19 mg/g), ‘Zardalu’ (12.43 mg/g) and‘Ratna’ (12.33 mg/g). The correlation between PPO activity(both catecholase and cresolase) and phenolic content was alsovery high (r=0.812), suggesting that the higher the PPO activity,the higher the level of phenolic compounds.

A strong inverse relation (r = -0.821) was observed betweenpolyphenol oxidase activity (both catecholase and cresolase)and malformation incidence. Genotypes with higher PPO activ-ity produced fewer deformed panicles than those with low PPOactivity. ‘Bhadauran’, which had the highest levels ofcatecholase and cresolase activity in its shoot tips, producedonly 1.10% malformed panicles (Table 1). In contrast, ‘TommyAtkins’, which had the lowest PPO activity, had the highestpanicle malformation incidence (53.75%) (Table 1). Higher PPOactivity was related to higher contents of phenolic compounds,which have been shown to provide resistance against diseases(Mayer and Harel 1979; Sharma et al. 1994).

ConclusionThese results indicate that PPO activity can be used to screenmango accessions for resistance to panicle malformation undernorthern Indian conditions. Further studies are needed to refinethe technique. Further studies are also needed into the inherit-ance of PPO activity to facilitate its use as a character that isused in selecting parental stock.

Based on the results for PPO activity, phenolic content andpanicle malformation, the 24 mango cultivars tested can be classi-fied into five groups, viz. highly resistant to panicle malformation(‘Bhadauran’ and H-8-1), moderately resistant (‘Dashehari’,‘Langra’, ‘Kurukkan’ and Fazli), susceptible (‘Sensation’, ‘Eldon’,’Rataul’, ‘Mallika’ and ‘Alphonso’), moderately susceptible (H-13-1, ‘Lal Sundri’, ‘Totapuri Red Small’, ‘Himsagar’, ‘Neelum’,‘Extrema’, ‘Zill’, ‘Edward’ and ‘Amrapali’) and highly susceptible(‘Tommy Atkins’, ‘Chausa’, ‘Zardalu’ and ‘Ratna’).

ReferencesDashhan, D.I.1987. Physiological studies on malformation of

mango panicles. Annals Agric. Sci. 32:565-575.Mayer, A.M. and E. Harel. 1979. Polyphenol oxidase in higher

plants: a review. Phytochem. 18:193-215.Nakamura, K., Y. Amano and M. Kagami. 1983. Purification and

some properties of a polyphenol oxidase from Koshu grapes .Am. J. Enol. Viticult. 34:122-127.

Pandey, R.M., M.M. Rao and R.K. Pathak. 1977. Biochemicalchanges associated with floral malformation. Sci. Hortic.11:37-44.

Pandey, S.N. 1998. Cultivars. Pp. 39–99 in Mango cultivation(R.P. Srivastava, ed.). International Book Distributing Com-pany, Lucknow, India.

Ram, S. 1991. Horticultural aspects of mango malformation.Acta Hortic. 291:235-252.

Ram, S. and V.K. Yadav. 1999. Mango malformation – A review.J. Appl. Hort. 1(1):70-78.

Sanchez-Ferrer, A., R. Bru, J. Cabanes, F.Garcia-Carmona.1988.Characterization of catecholase and cresolase activities ofgrape polyphenol oxidase. Phytochem. 27(2):319-320.

Sharma, R. R., H.C. Sharma and A.M. Goswami. 1994. Polyphe-nol oxidase activity and phenolic content pattern during shootdevelopment of grape (Vitis vinifera L.) in different growingseasons . J. Plant Biochem. Biot. 3(2):145-147.

Slinkard, K. and V.L. Singleton. 1977. Total phenol analysis:automation and comparison with manual methods. Am. J.Enol. Viticult. 28:49-55.

Valero, E., A. Sanchez-Ferrer, V. Varon and F. Garcia Carmona.1989. Evolution of grape polyphenol oxidase activity andphenolic content pattern during maturation and vinification.Vitis 28:85-95.

Vaughan, K.C. and S.O. Duke. 1984. Function of polyphenoloxidase in higher plants. Physiol. Plant. 60:106-112.

Wisseman, K.W. and C.Y. Lee. 1980. Polyphenol oxidase activityduring grape maturation and wine production. Am. J. Enol.Viticult. 31:201-211.

44 Plant Genetic Resources Newsletter, 2000, No. 124 Plant Genetic Resources Newsletter, 2000, No. 124: 44-50

A nested analysis to detect relationshipsbetween genetic markers and germplasm classesof durum wheatG. Figliuolo* and P. L. Spagnoletti ZeuliCentro Interdipartimentale per la Salvaguardia delle Risorse Genetiche Agrarie “Pierino Iannelli”, Università della Basilicata,Via N. Sauro, 85 – 85100 Potenza, IT. Email: [email protected]

SummaryA nested analysis to detectrelationships between geneticmarkers and germplasmclasses of durum wheatThe degree of differentiation betweendurum wheat cultivars and landrace ge-nomes due to breeding selection pres-sure is not known. A screening of a sub-core of landrace-derived lines and culti-vars was carried out with molecularmarkers (RFLPs and RAPDs) and seedstorage protein markers. Genetic diver-sity for each character within and be-tween germplasm classes was measuredusing both the percentage of polymor-phism and a sharing band genetic diver-sity index. Relationships among geno-types and the presence of subgroupswithin the population were revealed bycluster analysis. A nested cluster analysiswas conducted in order to identify thepresence of a hierarchical structure ofdiversity within a marker type. Althoughlandraces were more polymorph thancultivars, the two-germplasm categoriesdid not cluster separately and the degreeof dispersion of cultivars within landraceswas higher for RAPDs than for RFLPs orstorage proteins. RFLP was the onlymarker type able to detect two exter-nally isolated clusters instead of one.Within each RFLP cluster, cultivars sepa-rated as a more homogeneous grouponly with storage protein markers.RFLPs and storage proteins showed thebest ability to classify cultivars vslandraces. The differences of diversitybetween marker types and betweengermplasm category were highly signifi-cant. The modal values of genetic dis-tances were 0.6 for storage proteins, 0.163for RFLPs and 0.04 for RAPDs.

Key words: Durum wheat, geneticdiversity, RAPDs, RFLPs, storageproteins

ResumenUn análisis agrupado paradetectar relaciones entremarcadores genéticos y clasesde germoplasma de trigo duroNo se conoce el grado de diferenciaciónentre cultivares de trigo duro y genomasde variedades naturales por causa de lapresión de la selección genética. Se realizóun examen sistemático de un subgrupode linajes y cultivares derivados de var-iedades naturales, por medio de marca-dores de moléculas (RFLP y RAPD) ymarcadores de proteínas almacenadas enla semilla. Se midió la diversidad genéticade cada carácter dentro de y entre lasclases de germoplasma utilizando tanto elporcentaje de polimorfismo como uníndice de diversidad genética de bandacompartida. El análisis de conglomeradosreveló las relaciones entre genotipos y lapresencia de subgrupos dentro de la po-blación. Se realizó un análisis de conglom-erados agrupado para identificar la pres-encia de una estructura jerárquica de di-versidad dentro de un tipo de marcador.Aunque las variedades naturales eran máspolimórficas que los cultivares, las dos cat-egorías de germoplasma no formabanconglomerado separadamente y el gradode dispersión de los cultivares dentro delas variedades naturales era superior paralos RAPD que para los RFLP o las proteí-nas almacenadas. El RFLP era el único tipode marcador capaz de detectar dos con-glomerados externamente aislados enlugar de uno. Dentro de cada conglomer-ado RFLP, los cultivares se separabancomo grupo más homogéneo sólo conmarcadores de proteínas almacenadas.Los RFLP y las proteínas almacenadas re-sultaron ser los mejores criterios paraclasificar cultivares vs variedades natu-rales. Las diferencias de diversidad entretipos de marcadores y entre categorías degermoplasma eran muy significativas.Los valores modales de distancias genéti-cas eran 0,6 para proteínas almacenadas,0,163 para los RFLP y 0,04 para los RAPD.

RésuméDétection des corrélationsentre marqueurs génétiques etclasses du germoplasme deblé durum par méthode declassification emboîtéeLe degré de différenciation entre les gé-nomes de cultivars de blé durum et lesraces locales du à la pression de sélectionreste méconnu. Le criblage d’un sous-en-semble de lignées issues de races locales etde cultivars a été réalisé à l’aide de mar-queurs moléculaires (RFLP et RAPD) etde marqueurs de protéines de stockagedu grain. Les diversités génétiques intra-et interclasses ont été mesurée pourchaque caractère par le pourcentage depolymorphisme et l’index de diversitégénétique de ségrégation de bande. Descorrélations entre les génotypes et laprésence de sous-groupes dans la popula-tion ont été démontrées par une analysede classification. Une analyse de classifica-tion emboîtée a été réalisée afind’identifier l’existence d’une hiérarchisa-tion de la diversité pour un marqueurtype. Malgré un polymorphisme plusélevé pour les races locales que pour lescultivars, ces deux catégories de germo-plasme ne se différencient pas l’une del’autre et le degré de dispersion des culti-vars dans les races locales se montre plusélevé pour les RAPD que pour les RFLPou les protéines de stockage. Seul les mar-queurs RAPD sont capables de détecterdeux groupes marginaux au lieu d’un seul.Dans chaque groupe RFLP, les cultivarsségrègent en un ensemble plus ho-mogène que par l’utilisation des mar-queurs des protéines de stockage. Lesmarqueurs RFLP et des protéines destockage ont été les mieux à mêmed’établir une classification entre cultivarset races locales. Les différences de diver-sité entre les types de marqueurs et entreles catégories de germoplasme se sontrévélées très significatives. Les valeursmodales des distances génétiques ont étéde 0.6 pour les protéines de stockage, 0.163pour les RFLP et 0.004 pour les RAPD.

ARTICLE

IntroductionThe evolution of the durum wheat genepool, Triticum turgidum L.conv. durum (Desf.) MacKey (2n=4X=28), in the second half ofthis century, lies in the production and spreading of modernpopulations of derived lines (cultivars) from old, locally adapted,cultivated forms (landraces) (D’Amato 1989). Cultivars perform

better than landraces in terms of yield and quality traits and havean increased adaptation to high input agricultural systems. Theimprovement of the yield potential of durum wheat cultivarsfarmed in a less stringent climate expanded the traditional Medi-terranean area of cultivation (Auriau 1978; Baldy 1986).


From a microevolutionary point of view, landraces may haveexperienced little or no modern breeding selection pressure, thusmaintaining an adaptability to low input soils and to old agricul-tural practices. Plant germplasm such as landraces from differentgeographical origins could have evolved different gene complexesfavoring adaptation to local environmental conditions. In con-trast, modern cultivars largely share a common genepool becauseof common “good” genomes in their pedigrees.

For these reasons alleles should not be randomly distributedwithin the gene pool and, as consequence of a differentialpattern of variation between cultivars and landraces, the pres-ence of two distinct groups (landraces and cultivars) within thespecies is to be expected.

The genetic diversity available in this species has been stud-ied in large collections of germplasm using isozymes (Asins andCarbonell 1988), storage proteins (Damania et al. 1983), andquantitative morphological characters (Spagnoletti Zeuli andQualset 1987, 1990; Pecetti et al. 1992). Relationships betweentraits with adaptive value and origin were detected in somecases (Spagnoletti Zeuli and Qualset 1987, 1990; Pecetti et al.1992). However it is not clear whether the introgression of“good” genes from landraces to cultivars implied a linkage dragfor large portions of the genome fixed into cultivars followingbreeding, thus leading to a genome divergence of cultivars fromlandraces and for neutral alleles.

The availability of different classes of biochemical (storageproteins, isozymes) and molecular markers (RFLPs, RAPDs,microsatellites, etc.) makes the assessment of a direct measure ofgenetic variation possible. Appropriate genetic distance indicesmeasuring the amount of shared alleles can be used to detectgenome sharing within each germplasm class. The reordering ofgenetic distance indices by agglomerative clustering methodsbased on algorithms, such as single linkage analysis orunweighted pair group arithmetic averaging (UPGMA), can beemployed to visualize both the relationships between individu-als and the presence of one or more distinct groups (externallyisolated clusters) in the same population (Sneath and Sokal1973). In addition, set consistent cluster (Wong and Schaack1982) and discriminant analysis (Figliuolo and Spagnoletti Zeuli1997) using genetic distances as variables, can be used to quan-tify the discrimination ability by marker type.

The analysis of diversity based on the study of the hierarchyof different marker types within different germplasm classes,can give information about which stretches of DNA may beassociated with a phenotypic mutation common to a group ofindividuals (Templeton et al. 1992). Intraspecific trees obtainedwith germplasm lines from different geographic origins or withdifferent classes of germplasm, can promote a new criterion ofidentification of genetic resources useful for breeding purposes(Avise 1994; Wang et al. 1994; Paull et al. 1998).

Since the screening of large collections requires considerableeconomic resources, the use of a sub-core sample represents apreliminary step in the genetic analysis of wider collections.Preliminary studies using sub-core samples of genotypes, whoseclass membership is already known, could be used to discrimi-nate which marker class performs better in meeting the require-ments of germplasm management. Common questions of

genebank managers are how to re-allocate accessions to classesof interest when the information is not available or not correct atthe gene-bank (e.g. geographic origin) (Spagnoletti Zeuli andQualset 1987), and how to validate expected genepool member-ship (e.g. cultivars vs landraces).

The objectives of this work are:1) To study the genetic diversity of a sample of improvedItalian cultivars and a sub-core sample of morphologicallyhighly diverse germplasm accessions using seed storage pro-teins and molecular markers (RFLPs and RAPDs);2) To visualize the relationships among germplasm lines andthe hierarchy of diversity for different types of markers within aclustering trait (marker type) using a nested cluster analysis;3) To validate the a priori classification of cultivars vs accessionsby discriminant analysis of the whole set of loci for each differ-ent marker type.

Materials and methodsGenotypesThirty one different single-seed-derived lines from landraces ran-domly sampled from a durum wheat world core collection, ob-tained on the basis of the multivariate analysis of quantitativemorphological characters (Spagnoletti Zeuli and Qualset 1993),and fifteen durum wheat cultivars, 13 from Italy (Mariani andNovaro 1991) and 2 from the USA (‘Langdon’ and ‘Modoc’), wereanalyzed (Table 1). Chromosomal localization of cDNA cloneswas made using Chinese spring (CS) aneuploids of nullisomic-tetrasomic lines (NT) (Sears 1966) and disomic-substitutions linesof ‘Langdon’ durum wheat (Joppa and Williams 1977).

RFLP AnalysisGenomic DNA was extracted from leaf tissue of a single plant at2-3-tiller stage (Ellis et al. 1984). Following electrophoresisthrough 0.8% Agarose/TBE gel of EcoRI digested genomic DNA,the resulting fragments were transferred to Hybond-N nylonmembranes by alkaline blotting. The membranes were pre-hy-bridized and hybridized in 1M NaCl, 1% SDS, 50mM Na-Phos-phate at 65°C with 32P-labeled probes (megapriming DNA label-ing system) (Amersham) for 18 hours, washed sequentially with2X SSC, 0.1% SDS then 1X SSC, 0.5% SDS for 15 minutes each at65°C, and then exposed to X-ray films between two intensifyingscreens at -70°C for 6 days. Probes were 10 single and low copycDNA clones (0.4 - 1.3 Kb) randomly selected from a libraryobtained with mRNA of Triticum turgidum conv. durum Cv.Messapia (Figliuolo et al. 1993).

RAPD AnalysisTen primers were selected on the basis of their stability from 50oligo random 10 mer primers (Table 2b) (Promega Co., Madison,USA). Oligo sequences were defined by random assortment ofnucleotides with the constraint that the terminal basis of thesame primer were not homologous. Amplification reaction wascontained in 25 µl 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 1.5 mMMgCl2, 0.01% gelatin, 200 µM each of dNTP, 0.2 µM of primer,25 ng of genomic DNA and 2.5 U of termostable Taq DNApolymerase. DNA amplification was performed in a PerkinElmer Cetus DNA Thermal Cycler 480 programmed at 45 cycles


(1 min. at 94°C, 1 min. at 35°C, 2 min. at 72°C). Amplificationproducts were analyzed by electrophoresis in 2% agarose gelsand visualized by ethidium bromide staining. For data analysisfaint bands were not considered.

Protein Storage AnalysisStorage protein components were separated by electrophoresis.Acid-PAGE method was used for gliadines (Lafiandra andKasarda 1985) and SDS-PAGE for glutenins (Damania et al.1983). All components were scored for presence or absence.

Data AnalysisElectrophoregrams for each individual were scored and the pres-ence (1) or absence (0) of each band noted. The percentage ofpolymorphism (Graner et al. 1990) for each marker class wascalculated as the number of informative comparisons among allwheat individuals divided by the total number of comparisons.The percentage of polymorphism estimates the likelihood thattwo genotypes sampled from the population are diverse, withoutevaluating any degree of variation at one or more marker loci.

The “Dice” genetic diversity coefficient (GDij) (Nei and Li1979) was calculated as:

( )[ ]GD N N Nij ij i j= − +1 2 /

where GDij is the diversity coefficient between genotypes i and j;Nij is the number of bands common to i and j (shared bands); Niand Nj are the total number of bands for i and j respectively.Thus GDij=0 indicates identity between two lines, while aGDij=1 indicates complete diversity. Mean genetic dissimilarity(MGDij) of genotype i to a set j of genotypes was obtained byaveraging individual GDij estimates according to the followingformula:

where nj is the number of elements in set j.Cluster analyses were conducted with NTSYS-pc, version

1.80 (Exeter Software, Setauket, N. Y.) using as variables thediversity genetic indices (GDij) and the unweighted pair-groupmethod arithmetic average (UPGMA). The UPGMA methodwas used because among the parametric methods it is better atshowing the true cluster structure (Milligan 1980). The resultingclusters were expressed as dendrograms.

Table 1. Landraces and cultivars of triticum durum used for RFLPS, RAPDS and storage proteins analysis

LANDRACES CULTIVARS

# Country code Identifier # Name Pedigree

1 PRT (Portugal) PI 134935 1 LANGDON (Mindum/Carleton,Ld194)/2/Kapli/3/(Ld308,Heiti/Stewart/2/Mindum/Carleton)/4/Stewart/5/Carleton

2 ITA (Italy) PI 1579523 TUR (Turkey) PI 165181 2 MODOC (Tremez Molle Enano/2*Tehuacan 60/2/2*Zenati

Bouteille/Wells,D7069)/3/Leeds4 TUR PI 166470 3 APPIO Cappelli x (Gaviota x Yuma)5 TUR PI 1665746 TUR PI 166680 4 ARCANGELO Creso x Appulo7 TUR PI 1674848 TUR PI 173456 5 ANTHAS ((Yt54 x N10-B)BY2) Tc (Yt54 x N10-B) Tac125 Tc3 x

Ichnusa9 SYR (Syria) PI 18269910 MOR (Morocco) PI 184534 6 BRAVO Selection within a population of Creso11 PRT PI 18454412 TUN PI 185411 7 CRESO Yaktana54/Norin 10//Brevor///*2Cappelli-63/4/*3

Tehucan//5/Cappelli B14413 PRT PI 18574214 PRT PI 185768 8 DUILIO Cappelli x (Anhinga x Flaminio)15 SPA (Spain) PI 19098716 PRT PI 191627 9 GRAZIA M 6800127 x Valselva17 PRT PI 19186518 PRT PI 191979 10 MESSAPIA (Mex x Crane “S”) x Tito19 PRT PI 19214920 ETH (Ethiopia) PI 195093 11 NADIAN (Capeiti x Dauno III) x Beladi 11621 CYP (Cyprus) PI 21096722 EGY (Egypt) PI 220426 12 NORBA (Mex 10 x Crane) x (Creso x sel. Sicilia)23 IRN (Iran) PI 22531924 ITA PI 231358 13 TRINAKRIA B 14 x Capeiti 825 GRC (Greece) PI 26498926 HUN (Hungary) PI 290500 14 VALNOVA Ge 598 - Cappelli x {[Sel. F2 (Yt54-N10B) BY2]

Ld390, II 14587} x (Cappelli x Yuma)27 RUS (Russia) CItr 376628 RUS CItr 5014 15 VESPRO V.Z.1 x P.B.229 AZE (Azerbaijan) PI 5718630 UKR (Ukraine) PI 5760331 IND (India) CItr 8374

MGD GD nij ij j= ∑ /


If one marker type, using the whole set of lines,produced two externally isolated clusters during thefirst step, then a nested cluster analysis was carried outusing the set of lines included in each externally sepa-rated cluster (sub clustering). Such sub clustering wasconducted separately for marker types not used duringthe first step. This criterion was used to assess andvisualize the presence of a hierarchical structure ofgenetic diversity between one marker type and another.

In order to obtain estimates of correlations be-tween different marker types (cRFLPs, RAPDs andseed storage proteins), the diversity matrices based onthe “Dice” genetic distance were compared using theMantel matrix-correspondence test (Mantel 1967;NTSYS-pc instruction manual).

SAS statistical software (SAS 1989) was used tocarry out non-parametric discriminant analysis basedon the kth nearest neighbor method (PROC DISCRIMMETHOD=NPAR), using genetic distances within thegermplasm class. This analysis was relevant for testingdifferences in GDij indices between marker types withinand between germplasm groups and for evaluating theability (Kleinbaum et al. 1988) of each marker type toclassify the genetic distance indices into two pre-de-fined classes of germplasm (cultivars and landraces).

Results and discussionClusteringClusters obtained separately for each marker class(Fig.1), depict relationships among durum wheatlines. Cultivars did not form a clearly distinct clusterand their dispersion within and between landraceswas greatest with RAPDs (Fig. 1d). Landraces didcluster according to their geographic origin more withRFLPs than with storage proteins or with RAPD mark-ers (Fig.1).

Only with RFLP two externally separated clusterswere obtained, representing two groups within thewhole sample of genotypes. One of these clustersmainly includes germplasm accessions from theformer USSR (CItr 5014, CItr 3766, PI 57603), CItr8374 from India and PI 195093 from Ethiopia plusfive cultivars (‘Nadian’, ‘Grazia’, ‘Appio’, ‘Langdon’and ‘Modoc’) (Fig.1a).

Due to a lower degree of polymorphism, cultivarswere joining at a lower hierarchical level with the excep-tion of ‘Nadian’ which always joined the clusters at ahigh hierarchical level with each marker class (Fig.1).

A degree of diversity was observed for storageproteins only when the hierarchy first level was RFLPmarkers (Fig 1b). In this case cultivars separated intomore homogeneous groups within landraces both forthe first and second RFLP groups.

All clusters indicate that in terms of genetic rela-tionships among genotypes (shared traits), the im-proved germplasm did not form a differentiatedpopulation within the whole genepool. The more ho-

Fig. 1. Dendrogram for 46 durum wheat lines (15 cultivars and 31germplasm accessions) based on UPGMA method using geneticdiversity estimates from (a) RFLPs, (b) storage proteins nested withinRFLPs groups I and II, (c) storage proteins and (d) RAPDs.


mogeneous segregation of cultivars within landraces, when thegroups were analyzed for storage proteins (Fig 1b), could implythat selection pressure for storage proteins can be correlated toDNA fragments responsible for population subdivision, as visu-alized by the two distinct RFLP clusters.

The hierarchical analysis of diversity, by means of the nestedcluster analysis, improved the resolution of cultivar clustering.A cultivar dispersion within landrace durum wheat germplasmwas also observed by Autrique et al. (1996), using better genomesampling (39 RFLP clones). The cluster relationships of plantgenotypes by means of RFLPs and storage proteins reflected therestricted genetic background of some cultivars. ‘Cappelli’, thebase cultivar for ‘Capeiti’ (D’Amato 1989), is often present inthe pedigree of Italian cultivars because of its high semolinaquality. However some cultivars (‘Nadian’ and ‘Langdon’) hada distinct genetic makeup and clustered within germplasmaccessions. This pattern was also observed when RFLP markersdetected two clusters (groups) instead of one.

Percentage of PolymorphismRFLPs: Six out of ten (60%) cDNA clones, in combination with therestriction enzyme EcoRI detected polymorphism. The degree ofpolymorphism varied greatly among clones (Table 2a) and washigher among germplasm lines than among cultivars. The per-

centage of polymorphism was higher than 66.9% for three clones(pTdUBP1, pTdUBP7, and pTdUBP9) among landraces and fortwo clones (pTdUBP7 and pTdUBP9) among cultivars. For clonepTdUBP7 7 different restriction patterns were observed with 78.9%polymorphism among germplasm lines and 72.3% among culti-vars. The mean degree of polymorphism for RFLPs detected byrandom probes in durum wheat (27 %) was higher than thatdetected in cultivars of bread wheat (8 %) using 18 cDNA clonesmapped on chromosome 7 (Chao et al. 1989).

RAPDs: Amplified electrophoretic bands and a stable am-plification pattern were obtained with ten out of fifty primers(Table 2b). Eight primers produced polymorphic bands. Theaverage degree of polymorphism (RAPDs) per primer was gener-ally lower than the average degree of polymorphism (RFLPs) percDNA probe, although primers were more likely to be polymor-phic than cDNA clones. Four primers (UBP13, UBP19, UBP20and UBP21) showed a percentage of polymorphism higher than56.6% within germplasm lines, where the highest value (71.4%)was shown by UBP20 with 10 different amplification patterns.Among cultivars the highest value was (83.3%) with 6 differentpatterns (Table 2b).

Storage proteins: Gliadin and glutenin components werehighly polymorphic (100% polymorphism) and each genotypeshowed a unique electrophoretic pattern.

Table 2. Percentage of polymorphism, number of patterns and number of components per pattern for RFLPsusing 10 random cDNA clones (a) and for RAPDs using 10 random sequences 10 mer (b) in 46 durum wheatgenotypes.

(a)

Germplasm accessions (n°31) Cultivars ( n°15)

cDNA Kb Chromosomal RF (no.) Patterns RFLPs % RF (no.) Patterns RFLPs %clone location (no.) (no.)

pTdUBP 1 1.3 2B2D4D 13 6 66.9 13 4 37.1pTdUBP 2 1.0 5AL5BL5DL 5 3 47.1 5 1 0pTdUBP 3 1.1 5A5B5D 5 2 28.0 5 1 0pTdUBP 6 1.2 1A5B6D 2 1 0 2 1 0pTdUBP 7 1.0 2B2D 6 7 78.9 7 7 72.3pTdUBP 8 0.8 7AS7DS 5 3 37.4 5 2 34.3pTdUBP 9 1.0 7A7B7D 9 4 67.5 9 4 71.4pTdUBP 11 0.4 6A6B6D5D 5 1 0 5 1 0pTdUBP 117 0.7 1A1B1D 4 1 0 4 1 0pTdUBP 133 0.7 3A3B3D4B 2 1 0 2 1 0

(b)

Primer and Sequence Bands (no.) Patterns (no.) RAPDs % Bands (no.) Patterns (no.) RAPDs %

UBP3 (CGGGGTCGTA) 8 4 30.0 8 2 12.5UBP6 (CGTGAGCCAA) 7 2 12.5 7 2 12.5UBP10 (CTATCAGCGT) 1 1 0 1 1 0UBP12 (GACCTGCCTT) 4 4 39.6 4 4 35.0UBP13 (GAGGGCTCAT) 9 6 62.1 9 3 42.5UBP19 (GCACATGTTT) 5 6 56.6 5 5 45.0UBP20 (GCACCATCCT) 8 10 71.4 8 3 43.0UBP21 (GCAGCCGTAT) 7 8 70.1 7 6 83.3UBP22 (GCAGGTTGAA) 2 1 0 2 1 0UBP24 (GCCAGCCGCA) 5 2 32.3 5 1 0


Table 3 Genetic diversity estimates calculated for three marker classes (RFLPs, RAPDs, storage proteins,separately and pooled) and for each germplasm class (landraces, cultivars and pooled).

GERMPLASM MARKER GENETIC DISSIMILARITY

No.† Mean Mode Min MaxRFLPs 0.138 0.185 0 0.27

Landraces RAPDs 465 0.055 0.056 0 0.15(no. = 31) Storage Proteins 0.611 0.600 0.03 0.97

All 0.161 0.136 0.03 0.25

RFLPs 0.111 0.163 0 0.24Cultivars RAPDs 105 0.041 0.032 0 0.10(no. = 15) Storage Proteins 0.376 0.333 0.03 0.86

All 0.119 0.136 0.03 0.23

RFLPs 0.133 0.163 0 0.36Accessions RAPDs 570 0.053 0.044 0 0.15+ Cultivars(no.= 46) Storage Proteins 0.568 0.6 0.03 0.97

All 0.153 0.136 0.03 0.27

† Number of within germplasm class genotype combinations.

Genetic DiversityThe modal value of genetic diversity (mode of GDij) of the wholegermplasm sample was 0.60 for protein storage, 0.16 for RFLPsand 0.04 for RAPDs. The overall value for all the marker classeswas the same as the overall value in the two germplasm classes(Table 3). Genetic diversity indices were normally distributedonly for storage proteins and only within each single germplasmclass. Differences among marker types within and betweengermplasm classes were highly significant (protected LSD.05 ,P<0.001) (Table 3). Genetic distances calculated for each type ofmarker showed little or no correlation in the two germplasmclasses. A significant correlation (r = 0.35) was observed withincultivars between storage proteins and RFLPs.

The sample sets of within germplasm class genetic distances(GDij), after discriminant analysis, were properly classified intoaccessions by all marker types (more than 90% correctly classi-fied). However, RFLPs performed best by correctly classifyingthe 95.7% of genetic distances (Table 4). Storage proteins werebest at classifying GDij into cultivars (54% correct classifica-tion), but not one genetic distance was properly classified byRAPD markers (Table 4).

Genetic diversity indices (GDij) have the ability to detect thedegree of variation in terms of the overall proportion of notshared bands between two genotypes. For this reason they areinversely correlated to the average number of genomes thatindividuals within a population share because of common an-cestors. In addition, genetic diversity indices are reliable data forgenetic diversity studies. They are additive values within thedissimilarity matrix sample space which enable the estimate ofthe average level of genetic diversity of a population and theidentification of components affecting their variation. The esti-mates of genetic distances appear to be more severely affected bythe number of loci sampled than by the number of individuals(Nei 1978).

Genetic diversity indices for each pairwise comparison weresorted (data not shown) and the highest dissimilarity index forRFLPs (GDij=0.36) was between PI190987 and ‘Nadian’. The

highest value for RAPD (GDij=0.15) was observed between PI191627 and PI 220426. For gliadin and glutenin componentsgenotype PI 220426 combined with lines PI 182699 and PI231358, and PI 225319 combined with PI 185742 and CItr 3766generated indices higher than GDij=0.95.

The unimproved germplasm was significantly more variablethan cultivars. Melchinger et al. (1994) detected an average ge-netic similarity (GS) of 0.85 (GD=0.15) in winter barley andGS=0.84 (GD=0.16) in spring barley using 48 single copy DNAclones. Sielder et al. (1994) detected a GD=0.08 in winter wheatgermplasm, a GD=0.11 in spring germplasm (T. aestivum) and aGD=0.10 in T. spelta using 58 probe/enzyme combinations.Therefore, the RFLPs average GD estimates in T. durum resultedhigher than in T. aestivum L. and T. spelta L., and lower than inHordeum vulgare L..

Protein storage markers and RFLPs were more informativethan RAPDs for their ability to classify two pre-definitegermplasm classes (Fig. 1a and b; Table 4). The inability ofRAPDs to classify the genetic diversity indices in cultivars couldimply that this class of markers has been less influenced byartificial selection. RAPDs could be useful for detecting poly-morphism in germplasm which is very homogeneous for knowngenetic markers. Storage protein markers classified the twogermplasm classes better than RFLPs and were able, with justtwo biochemical analyses, to identify all the genotypes asunique. The limit of storage proteins is to report variation onchromosomes one and six within gene families. RFLP markers

Table 4. Percentage of genetic diversity indices (% ofDij) correctly classified in each germplasm class,following discriminant analysis, by each marker type.

(% of D ij) Landraces Cultivars

Storage Proteins 94.4 54.3RFLPs 95.7 35.2RAPDs 90.7 0All 94.8 33.3


should be preferred when exploring genetic diversity because oftheir stable genetic basis, their moderate level of polymorphism,their codominant nature and wide genome distribution.

ConclusionsIn this study it has been shown that the durum wheat genepool(unimproved and improved) is largely a unique population, andartificial selection in the second half of the century has notsufficiently differentiated germplasm lines from cultivars atgenomic level. The hierarchical analysis of diversity using anested cluster analysis improved the resolution of cultivar clus-tering for storage proteins. A nested cluster analysis, instead ofonly a merely horizontal comparison of different classes ofmarkers, can show some degree of hierarchy of the geneticdiversity that could be useful for better managing genetic re-sources in genebanks.

Our results confirm the usefulness of molecular marker datato score genetic variation within durum wheat for germplasmclassification and breeding purposes, as well as the existence ofa moderate level of genetic diversity in durum wheat cultivars.The identification of genetically distinct cultivars is useful forbreeding programs. An “a-priori” classification of durum wheatgermplasm based only on its breeding history would be ineffi-cient if the goal were to expand genetic variation useful forwheat improvement.

AcknowledgmentsThis work was supported by a grant from Ministero delle RisorseAgricole, Alimentari e Forestali (IT). We thank professor S.Benedettelli for his useful suggestions and professor A. Blancofor providing seeds of durum wheat Italian cultivars.

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RésuméUtilisation du germoplasme deriz conservéDans une étude menée en 1995 auprèsdes utilisateurs du germoplasme de rizissu de la Collection de la banque de gèneinternationale de riz à l’IRRI et concer-nant la période 1989-1994, 48 personnesinterrogées sur les 200 sollicitées ontdécrit l’évaluation et l’utilisation de pr-esque 400 échantillons. Il apparaît queseul un petit nombre de ces échantillonssert directement à l’amélioration du riz.La plupart sont évalués pour les stressbiotiques et abiotiques. La recherche agénéré 59 publications.

Use of conserved rice germplasmG.C. Loresto, E. Guevarra and M.T. Jackson*Genetic Resources Center, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines.Tel: (63-2) 845-0563; Fax: (63-2) 845-0606; E-mail: [email protected]

SummaryUse of conserved ricegermplasmIn a 1995 survey of users of germplasmfrom the International Rice GenebankCollection at IRRI between 1989 and1994, 48 respondents (from about 200surveyed) reported evaluation and useof almost 4000 samples. Only a smallnumber were used directly for rice im-provement. Most were evaluated for bi-otic and abiotic stresses. The researchgenerated 59 publications.

Keywords: Conservation, ex situ,Oryza, rice

ResumenUso de germoplasma de arrozconservadoEn una encuesta realizada en 1995 entreusuarios de germoplasma de la Colec-ción del Banco de Germoplasma Interna-cional de Arroz del IRRI entre 1989 y1994, se recibieron 48 respuestas (entreunos 200 encuestados) sobre evaluacióny uso de casi 4000 muestras. Sólo unaspocas se utilizaron directamente para lamejora del arroz. En la mayoría se eval-uó el estrés biótico y abiótico. La investi-gación dio lugar a 59 publicaciones.

SHORT COMMUNICATION

IntroductionCrop breeders draw on genebanks to raise yield potential, im-prove nutritional quality and tackle a broad range of challengesto agricultural productivity (Plucknett et al. 1987). Genebanksbackstop breeding programmes by regularly supplying newmaterials for specialized breeding pools. When the desired genesare not found in elite breeding lines, scientists turn to conservedgermplasm that comprises farmers’ or landrace varieties, oldcultivars and wild species.

In 1977 the International Rice Research Institute (IRRI)established the International Rice Genebank (IRG) committedto the long term-conservation of rice genetic resources. Today,over 107 000 accessions of Oryza are conserved in the genebank.These are duplicate collections of the national programmesfrom more than 110 countries in Asia, Africa, Oceania/Pacific,and North and South America.

The value of conserving rice genetic resources may be mea-sured in how these genetic resources are used in crop improve-ment, and eventually in a released variety. The studies byEvenson and Gollin (1994) indicated that the rice germplasmcollection at IRRI had a direct impact on the international flowof improved rice varieties, which in turn was directly associatedwith an increase in varietal releases and yield growth. Theyconcluded that the collection, preservation and cataloguing ofrice germplasm directly and unambiguously led to a higherproduction of rice around the world, and hence played a signifi-cant role in feeding millions of people.

The use of rice genetic resources is not limited to plantbreeding or crop improvement. Each year, thousands ofgermplasm samples are sent out for some kind of evaluationand research by users (Chang 1992). Documentation of thereported results, however, is scarce (Jones 1984). Many of theaccessions requested from the International Rice Genebank areused in a wide variety of research projects that expand ourunderstanding of the rice genepool and its value.

Survey of rice germplasm usersIn October 1995, we began a survey among scientists in sevencountries who received germplasm from 1989 to 1994. The objec-tives were to document how the requested germplasm was used,what traits were identified and used in breeding or crop im-provement, what methodologies were developed, and what othercontributions were made to rice science. More than 200 letterswere sent to recipients of germplasm in Bangladesh, Indonesia,Japan, Korea, Thailand, UK and USA; 48 users responded to ourqueries (Table 1).

Use of germplasmScientists who received conserved germplasm generated 59 pub-lications. These scientists used 3921 samples of cultivated rice(some 3800 distinct accessions) and 139 samples of wild species(105 distinct accessions) in various research activities. Recipi-ents used about 130 accessions of cultivated rice for crop im-provement. The traits transferred were resistance to brownplanthopper and bacterial leaf blight, low amylose, seedlingvigour, germination ability at low temperature, waxy endosperm,smooth hulls, and milling and cooking quality. Breeders areusually interested in using only a small fraction of the totalgermplasm at any one time to meet immediate and often press-ing objectives (Gill 1989). To use germplasm effectively, breedersneed variability available in an agronomically desirable back-ground.

Most of the accessions requested were used in the evaluationfor biotic and abiotic stresses (Table 1). For example, in Thai-land, plant breeders evaluated 122 accessions for grain qualityand found one (Acc. 9032) that could possibly be released tofarmers directly without further improvement. Researchers fromthe University of Wales-Bangor in the UK evaluated more than2300 accessions for cold tolerance, with the objective of using thevarieties identified as parents in a breeding programme in Nepal.The data generated will be stored in a database for other breeders


Table 1. Use of conserved germplasm from the International Rice Genebank Collection at IRRI between 1989and 1994.

Institution/ No. of Purpose Traits Traits Varieties releasedcountry samples identified† transferred and contributions

to rice science‡

BangladeshBangladesh 69 Evaluation, BPH, WBPH, callus Acc. 55814 used in NoneRice Research breeding induction & plant crossing programmeInstitute (BRRI) regeneration (Acc. 55814)BRRI 123 Evaluation BPH resistance BPH resistance gene NoneBRRI 4 Research Bacterial blight resistance None Plan to use as

differential varietiesin Bangladesh

BRRI 8 Research Resistance to tungro virus No publication(PhD thesis)

University of 37 Research Work on holdChittagong

IndonesiaInter University 7 Research AwaitingCenter for sponsorshipBiotechnologySoegijapranata 57 Re-introduced None NoneCatholic University to farmer’s field

JapanChugoku National 19 Evaluation RTSV, GLH (49)Agricultural & researchExperiment StationGifu University 27 Research Shade tolerance Deriphat-SDS page

technique forchlorophyll protein

Hokkaido 20 Research Cold tolerance at No publicationUniversity booting stageKagawa 35 Research Nitrate reductase (2)University Nitrite reductaseKobe University 16 Research Genetics of BPH resistance No conclusive resultsKyushu University 5 Research Comparison on floating ability No publicationNagoya University 1 Research Allergenic protein (1)Nagoya University 7 Research Salt tolerance Physiology and

morphology of rootsystem

National Agricultural 4 Breeding Low amylose Low amylose NoneResearch CenterNational Institute of 62 Research Blast resistance RFLP/RAPD,Agrobiological experiment terminatedResourcesUniversity of Tokyo 248 Research Rooting ability Used by graduate

student

KoreaSeoul National 2 Research Highly resistant Physiological plantUniversity to rice blast pathology, (46)Yung Nam Crop 39 Evaluation, Seedling vigour (4 acc.), Seedling vigour, NoneExperiment breeding, BPH resistance (1), germination abilityStation, RDA research germination ability at

low temperature (1)

ThailandDepartment of 151 Breeding, BPH (6 acc.),SB resistance BPH resistanceAgriculture (DA) evaluation (1 acc.), BLB resistance (Acc. 237), BLB

(2 acc.), grain char. (2 acc.) resistance (Acc. 611)grain quality (1 acc.) Acc. 9032 may be

released to farmersDA 8 Breeding BPH resistanceDA 8 Evaluation None None NoneDA 2 Evaluation Gall midge resistance Biotype study of rice

gall midgeDA 3 Research BPH-resistant check

United KingdomIACR-Rothamsted 147 Research Root growth in response Root physiology in rice,

to mechanical impedance screening method forroot elongation rate


Institution/ No. of Purpose Traits Traits Varieties releasedcountry samples identified† transferred and contributions

to rice science‡

John Innes Centre 1 Research Susceptible to isolates of Used for agroinoculation Molecular biology &two RTV & all biotypes of rice tungro bacilliform variation of the twoof Nephotettix virescens virus tungro viruses

University of 13 Research To confirm work of For further studyCambridge Yeo & FlowersUniversity of 4 Research Artificial symbiotic association Research group phasedDundee between rice & cyanobacteria outUniversity of 64 Research Biotechnology, (3-7,9-16,Nottingham 20-22,25,26,30-40,47,50-52)University of 21 Research Seed storage, seedReading germination, & seed

production; (17, 18, 19)University of 116 Research Salinity Results were used (23, 24, 27, 28, 29)Sussex & evaluation by IRRI breedersUniversity of 1 Research Ethylene responsive Tissue probed for ethyleneWales-Aberystwyth receptor proteinsUniversity of 2,363 Evaluation Superior cold tolerance Varieties identified will Replicable method forWales-Bangor be used as parents in screening cold tolerance.

a future breeding Useful as a databaseprogramme in Nepal for other breeders

University of 6 Research Long, thick roots Hydroponics root screeningWales-Bangor

United StatesPrivate citizen 11 Evaluation, Long grain size

researchBrigham Young 4 Research Observed wax crystalUniversity done at IRRI patternsCalifornia Cooperative 96 Breeding Still being evaluated forRice Research stem rot & sheath spotFoundation, Inc. resistanceCalifornia Cooperative 6 Breeding Waxy endosperm (Acc. Waxy endosperm, Many advanced lines forRice Research 76311); early maturity, smooth hull (Acc.76311); cooking quality.MethodologyFoundation, Inc. cooking & milling quality, milling & cooking quality developed: cooking quality

short grain (Acc. 76312) (Acc. 76312) screeningCornell University 167 Research BLB-R locus, Xa21 Genetic mapping, gene

tagging, evaluation of geneticdiversity using molecularmarkers, RFLP.(8,41,45,48,53,54,59)

Michigan State 5 Research Proteinase inhibitor II, Genes transferred were Varieties were used to trainUniversity & training bar gene proteinase inhibitor II Rockefeller Foundation

and bar gene students in geneticengineering

The Scripps Research 13 Research Characterization of riceInstitute, California tungro virus. Partial

desiccation of matureembryo-derived calli toimprove indica riceregeneration. Improvingfrequency of plantregeneration. (43, 44, 53)

University of 24 Research Drought-induced Extraction of seedlingCalifornia proteins (8 accs.) dehydrins (6 accs.)University of 4 Research Genetic engineeringWashington using AgrobacteriumVirginia Polytechnic 20 Research To evaluate photosynthesis, Use in physiologicalInstitute & State leaf growth and water use studies of a PhD thesis. (42)UniversityWashington State 5 Research Explant used to develop Experiment not successfulUniversity embryogenic calliUSDA-Maryland 4 Research Rice blast reaction Virulence screening

methods refinedUSDA-Idaho 3 Conservation

& distributionto researchers

† BPH = brown planthopper, WBPH = white-backed planthopper, GLH = green leafhopper, BLB = bacterial leaf blight, SB = stem borer, RTSV= rice tungro spherical virus, RTV = rice tungro virus, RFLP – restriction fragment length polymorphism, RAPD = random amplified polymor-phic DNA.‡ The number in parentheses refers to the publication in the list of publications generated.


to use. The group also developed a methodology for screeningcold tolerance, a vital tool in a breeding programme for this trait.Likewise, another group of researchers worked on 24 accessionsof cultivated rice and 11 wild species to understand the mecha-nism of salinity tolerance, and evaluated germplasm accessionsand breeding lines. IRRI breeders used the results of these stud-ies in the breeding programme for salinity tolerance. Plant breed-ers in the USA identified waxy endosperm, smooth hulls, andmilling and cooking quality from two accessions. They devel-oped a methodology to screen and identify advanced lines andvarieties for cooking quality.

Use of conserved germplasm is not limited to evaluation andcrop improvement. Experimental biologists and biotechnologistsalso use this conserved germplasm to advance rice science. Insome cases, germplasm was used solely in research or for teach-ing purposes, as at Michigan State University, where the fiverice varieties requested were used to train students in geneticengineering. At Virginia Polytechnic Institute and State Univer-sity, a PhD student used the requested accessions to evaluatephotosynthesis, leaf growth and water use of rice.

ConclusionsThe value of conserved germplasm thus lies not only in how ithas contributed to varietal improvement but also in rice scienceitself by expanding understanding of the physiological, mor-phological and genetic diversity of the crop and adaptation toits environment. The distributed germplasm has contributedimmensely to knowledge on the physiology and morphology ofthe root system, physiology of plant resistance to rice blast, abiotype study of rice gall midge and other areas. It also hasenhanced the use of molecular tools and biotechnology in evalu-ating crop diversity and improving the crop. The research workof Ellis et al. (1993) at the University of Reading, UK, and at IRRI,on seed physiology has contributed to the production of high-quality seeds for ex situ conservation, especially for long-termstorage. Moreover, screening methods for different biotic stresseshave been refined and further developed. These tools are help-ing rice scientists to understand better the nature of geneticdiversity in rice. In the future, they will also help produce newvarieties and make the conservation of rice genetic resourcesmore efficient.

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Publications generated using ricegermplasm from the International RiceGenebank Collection at IRRI, requestedfrom 1989 to 1994 (reference numbersare cited in Table 1).1. Adachi, T., A.M. Alvarez, N. Aoki, R. Nakamura, V.V. Garciaand T. Matsuda. 1995. Screening of rice strains deficient in 16-kDa allergenic protein. Biosci. Biotech. Biochem. 59:1377-1378.2. Barlaan, E.A. and M. Ichii. 1996. Genotypic variability innitrate assimilation in rice. Pp. 434-440 in Rice Genetics III.Proceedings of the Third International Rice Genetics Sympo-sium (G.S. Khush, ed.). International Rice Research Institute,Manila, Philippines.3. Blackhall, N.W., M.R. Davey and J.B. Power. 1994. Applica-tions of protoplast technology, Section A: Fusion and selectionof somatic hybrids. Pp. 41-48 in Plant Cell Culture: A PracticalApproach. 2nd Edition (R.A. Dixon and R.A. Gonzales, eds.).IRL Press, Oxford.4. Blackhall, N.W., M.R. Davey and J.B. Power. 1994. Isolation,culture and regeneration of protoplasts. Pp. 27-39 in Plant CellCulture: A Practical Approach. 2nd Edition (R.A. Dixon andR.A. Gonzales, eds.). IRL Press, Oxford.5. Blackhall, N.W., P.T. Lynch, J.P. Jotham, M.R. Davey andE.C. Cocking. 1993. A general procedure for the initiation of cellsuspension cultures of wild rice. P. 194 in Proceedings of theSixth Meeting of the Rockefeller International Program on RiceBiotechnology, Chiang Mai, Thailand, 1-5 February 1993.6. Blackhall, N.W., R.P. Finch, J.B. Power, E.C. Cocking andM.R. Davey. 1995. Flow cytometric quantification ofelectroporation-mediated uptake of macro-molecules into plantprotoplasts. Protoplasma 186:50-56.7. Blackhall, N.W., R.P. Finch, M.R. Davey and E.C. Cocking.1990. Flow cytometry to assess the efficiency of electroporation-mediated delivery of macromolecules to plant protoplasts. P. 21in Abstracts, International Conference on Electroporation andElectrofusion. Marine Biological Laboratory, Woods Hole, Mas-sachusetts, USA. October 1990.8. Causse, M., T.M. Fulton, Y.G. Cho, S.N. Ahn, K. Wu, J. Xiao,J. Chunwongse, Z. Yu, P.C. Ronald, S.B. Harrington, G.A. Sec-ond, S.R. McCouch and S.D. Tanksley. 1994. Saturated molecu-lar map of the rice genome based on an interspecific backcrosspopulation. Genetics 138:1251-1274.9. Cocking E.C., P.T. Lynch, N.W. Blackhall and M.R. Davey.1991. Rice protoplasts genetic manipulations. P. 49 in Abstracts,Fifth Annual Meeting of the Rockefeller International Programon Rice Biotechnology.10. Cocking, E.C., N.W. Blackhall, B. de Touchet, N.B. Jelodar,J.B. Power and M.R. Davey. 1993. Studies on rice somatichybridisation. XV International Botanical Congress, Tokyo, Au-gust 28-September 3, 1993.11. Cocking, E.C., P.T. Lynch, N.W. Blackhall, J. Jones, J.P.Jotham, G.S. Khehra, S.L. Kothari, S-H. Lee, K. Tang, B. deTouchet, P.S. Eyles and M.R. Davey. 1993. Rice genetic manipu-lations: use of plants regenerated from protoplasts. Pp. 120-121


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Molecular analysis of variability in Podophyllumhexandrum Royle – an endangered medicinal herb ofnorthwestern HimalayaK.D. Sharma¹*, B.M. Singh¹, T.R. Sharma², Meenu Katoch¹ and S. Guleria¹¹ Biotechnology Centre, Himachal Pradesh Krishi Vishvavidyalaya, Palampur 176062, India. Tel/Fax: +91 01894 30371;Email: [email protected]² National Research Centre on Plant Biotechnology, New Delhi 110012, India

SummaryMolecular analysis of variabilityin Podophyllum hexandrumRoyle – an endangeredmedicinal herb of northwesternHimalayaGenetic diversity in Podophyllum hexan-drum populations from HimachalPradesh, a part of the northwestern Hi-malayan region was studied by usingprotein and RAPD markers. Morpholog-ically, P. hexandrum from northwesternHimalayan region could be divided intofour variants having 1, 2, 3 and 4 leavesrespectively. SDS-PAGE of root proteinextracts from 30 P. hexandrum plants col-lected from Chamba and Kullu regionsproduced 23 protein markers, of which16 were polymorphic. The plants weregrouped into two broad groups (A andB) at 65% level of genetic similarity usingUPGMA. Group B was again subdividedinto three subgroups (B1-B3) having ge-netic similarity between 79 and 82%.RAPD analysis of genomic DNA of 30selected plants using seven 10-mer prim-ers revealed high inter- and intra-popu-lation genetic diversity. A total of 76 DNAmarkers, all of them polymorphic, wereproduced. Coefficient of similarity be-tween groups ranged between 0.25 to0.75, and 73.3% of the plants could bedivided into 6 groups (I-VI), of which 4groups (I, II, III and VI) contained plantsfrom Chamba and one group (IV) fromKullu. Group V included plants fromboth the locations. Protein markerscould not delineate P. hexandrum popula-tions into region-specific groups. RAPDmarkers, however, formed distinct clus-ters of plants from Chamba and Kulluregions. RAPD was found to be moresuitable for the elucidation of genetic di-versity in P. hexandrum compared withprotein analysis.

Key words: Genetic diversity, Podo-phyllum hexandrum, polymorphism,protein markers, RAPD

ResumenAnálisis molecular devariabilidad en el Podophyllumhexandrum Royle, una hierbamedicinal amenazada delHimalaya noroccidentalSe estudió la diversidad genética de laspoblaciones de Podophyllum hexandrumde Himachal Pradesh, parte de la regióndel Himalaya nororiental, utilizandomarcadores de proteínas y RAPD (ADNpolimorfo de amplificación aleatoria).Morfológicamente, el P. hexandrum de laregión del Himalaya noroccidental po-dría dividirse en cuatro variantes de 1, 2,3 y 4 hojas respectivamente. SDS-PAGEde extractos de proteínas de raíces de 30plantas P. hexandrum recogidas en las re-giones de Chamba y Kullu produjeron23 marcadores de proteínas, de los que16 eran polimórficos. Las plantas se rep-artieron en dos grandes grupos (A y B) alnivel del 65% de similaridad genética us-ando UPGMA. El grupo B se subdividióa su vez en tres subgrupos (B1-B3) consimilaridad genética entre el 79 y el 82%.El análisis RAPD del ADN genómico de30 plantas selectas usando siete 10-mersprimers reveló una alta diversidadgenética entre poblaciones y dentro decada población. Se produjeron en total 76marcadores de ADN, todos ellospolimórficos. El coeficiente de similaridadentre grupos osciló entre 0,25 y 0,75, y el73,3% de las plantas pudieron dividirseen 6 grupos (I-VI), 4 de los cuales (I, II, IIIy VI) contenían plantas de Chamba yuno (IV) de Kullu. El grupo V conteníaplantas de ambos lugares. Los marcado-res de proteínas no pudieron delinearpoblaciones de P. hexandrum en grupospor regiones específicas. Los marcadoresRAPD, por el contrario, formaron gru-pos netos de plantas de las regiones deChamba y Kullu. Se concluyó que la téc-nica RAPD era más adecuada para es-clarecer la diversidad genética del P. hex-andrum, en comparación con el análisisproteínico.

SHORT COMMUNICATION

RésuméAnalyse moléculaire de lavariabilité chez Podophyllumhexandrum Royle, une plantemédicinale menacée du Nord-Ouest de l’HimalayaLa diversité génétique de populations dePodophyllum hexandrum issues de Him-achal Pradesh, région du Nord-Ouest del’Himalaya a été étudiée en utilisant desmarqueurs protéiques et RAPD. AuNord-Ouest de l’Himalaya, P. hexandrumse répartit en quatre variants mor-phologiques possédant respectivement1, 2, 3 et 4 feuilles. La technique SDS-PAGE sur des extraits protéiques de ra-cine de 30 plants de P. hexandrum col-lectés dans les régions de Chamba etKullu a révélé 23 marqueurs protéiquesdont 16 étaient polymorphiques. Lesplantes ont été classées en deux grandsgroupes (A et B) avec un coefficient desimilarité génétique de 65% par la méth-ode UPGMA. Le groupe B a été subdiviséen trois sous-groupes (B1-B3) ayant uncoefficient de similarité génétique de 79%à 82%. L’analyse RAPD de l’ADNgénomique des 30 plantes sélectionnéespar l’utilisation d’amorces de 10 mer arévélé une forte diversité génétique in-ter- et intrapopulation. Un total de 76marqueurs ADN, tous polymorphiques,a été produit. Le coefficient de similaritéentre les groupes a varié entre 0.25 et0.75. 73.3% des plantes ont pu être dis-tribuées en 6 groupes (I-VI), dont 4 (I, II,III et VI) rassemblaient les plants issus deChamba et un (IV) ceux de Kullu, legroupe V incluant des plantes des deuxsites. Les marqueurs protéiques n’ontpas permis la détermination des popula-tions de P. hexandrum en groupes ré-gion-spécifiques. Cependant les mar-queurs RAPD ont distingué des ensem-bles de plants provenant des régionsChamba et Kullu. Les marqueurs RAPDse révèlent être plus adaptés pourl’explication de la diversité génétique deP. hexandrum que l’analyse protéique.

IntroductionIndia – a country of immense biotic wealth – has more than 7000species reportedly used for medicinal purposes (Groombridge1992) most of which are being exploited recklessly for the extrac-tion of drugs. It will be prudent to study species of indigenousmedicinal plants at genetic and molecular levels for efficient

conservation and management of genetic diversity. Study of inter-and intraspecific variation at the molecular level provides anefficient tool for taxonomic and evolutionary studies and fordevising strategies to protect genetic diversity of species. Geneticvariability also can be exploited to select useful genotypes that


could be utilized as cultivars to avoid batch-to-batch variation inextraction of standard drugs. Recent global emphasis on exploita-tion of herbal resources and instances of patenting of developing-country plants by developed countries emphasize the need togenerate databases on indigenous medicinal plants which can beused for future reference.

Himalayan mayapple (Podophyllum hexandrum Royle), an en-dangered medicinal herb, grows wild in the interior Himalayanranges of India. It is recognized for its anti-cancer properties.The rhizomes and roots of P. hexandrum contain anti-tumorlignans such as podophyllotoxin, 4’-demethyl podophyllotoxinand podophyllotoxin 4-o-glucoside (Tyler et al. 1988; Broomheadand Dewick 1990). Among these lignans, podophyllotoxin ismost important for its use in the semisynthesis of anti-cancerdrugs etoposide and teniposide (Issell et al. 1984). Podophyllo-toxin content in Himalayan mayapple is high (4.3%) comparedwith other species of Podophyllum, notably P. peltatum (0.25%), themost common species in the American subcontinent (Jacksonand Dewick 1984).

The population size of P. hexandrum in Garhwal Himalayas isvery low (40-700 plants per location) and is declining each year.Some of the populations in certain pockets have virtually disap-peared owing to anthropogenic activities and overexploitation(Bhadula et al. 1996). Thus, there is a need to conserve geneticdiversity of this prized medicinal plant which may become extinctif its reckless exploitation continues. Considerable variation inmorphological characters such as plant height, leaf characteris-tics, fruit weight, seed weight and colour, etc. and in biochemicalcharacters such as podophylloresin and podophyllotoxin contentin rhizomes has been reported in P. hexandrum plants from GarhwalHimalayas (Bhadula et al. 1996; Airi et al. 1997; Purohit et al. 1999).At least four distinct morphological variants – i.e. having 1, 2, 3and 4 leaves – have been reported (Purohit et al. 1998). Polypeptideprofiles in seeds of these variants indicate that these may begenetically distinct from each other. Polypeptide patterns andeasterase isozyme analysis have indicated the existence of highinter- and intrapopulation variation in P. hexandrum populationsfrom Garhwal Himalaya (Bhadula et al. 1996). However, morpho-logical and protein markers are influenced by the stages of plantgrowth as well as environmental factors and hence may giveerroneous results. DNA markers such as RFLPs (Botstein et al.1980) and RAPD (Williams et al. 1990; Kazan et al. 1993) on theother hand are quite stable and highly polymorphic in nature.RFLP markers are less polymorphic, more expensive andlabourious compared with RAPD. To our knowledge, there is nopublished information on the use of RAPD markers for the char-acterization of genetic diversity in P. hexandrum.

The objectives of the present study were to use protein andRAPD markers for the molecular characterization of geneticvariability in the natural populations of P. hexandrum from thenorthwestern Himalayan region.

Materials and methodsPlant materialThirty plants of P. hexandrum collected from Chamba (location:Khajjiar) and Kullu (location: Jalori Pass) districts of HimachalPradesh were selected for protein and RAPD assay. These two

locations are situated at 2400 and 2900 m above mean sea level.The plants from Chamba district were designated as CH59,CH26, CH44, CH8, CH34, CH57, CH39, CH61, CH30, CH46,CH63, CH62, CH25, CH18, CH28, CH69, CH9, CH10, CH65,CH70, CH51 and CH11 whereas those from Kullu were desig-nated as J43, J16, J10, J35, J13, J47, J18 and J45.

Protein extraction and electrophoresisAcidic proteins were extracted from freshly harvested roots of P.hexandrum in 250 mM sodium acetate buffer (pH 5.2). The pro-teins were precipitated in 40% TCA at –20ºC for 30 minutes anddissolved in sample buffer (0.0625M Tris HCl pH 6.8, 2% SDS,5% β-mercaptoethanol, 10% sucrose and 0.002% bromophenolblue). Concentration of proteins in each sample was determinedby using Bradford method (Bradford 1976) and the final con-centration was adjusted to 2 µg protein/µl of sample buffer.Protein extract from each P. hexandrum plant was boiled in a waterbath for 5-10 min and 40 µg of protein per sample was loaded onsodium dodecyl sulfate-polyacrylamide gel containing 12.5%polyacrylamide (Lammeli 1970) for electrophoresis. The gelswere stained with coomassie blue and visualized in white fluo-rescent light.

DNA extraction and PCRTotal genomic DNA was extracted from young leaves of P.hexandrum using CTAB method (Saghai-Maroof et al. 1984). Afterprecipitation, DNA was suspended in 100 µl of T.E. buffer (pH8.0) for use in PCR. PCR was carried out in 25 µl reaction mixturecontaining 1-1.5 unit Taq DNA polymerase, 0.8 mM dNTP mix,10mM Tris HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 5 pMprimer (Operon Technologies Inc. USA) and 25-50 ng of genomicDNA. Amplification was performed in a PCR machine (DNAengine 200, MJ Research) with the following temperature profiles:94ºC for 5 min for denaturation of genomic DNA, 37ºC for 1 minfor primer annealing and 72ºC for 1 min for primer extension.Forty additional cycles were performed at 95ºC for 1 min, 37ºC for1 min and 72ºC for 2 min. The last cycle was run at 72ºC for 5 min.

The amplification products were resolved in 1.4% agarose gel(1x Tris-acetate-buffer) followed by ethidium bromide staining andvisualization in UV light for photography (Sambrook et al. 1989).

Recording of data and analysisRAPD as well as protein profile data from each plant wererecorded as binary system where 1 and 0 indicated the presenceand absence of a particular band. The data were analyzed usingNumerical Taxonomy System of Multivariate StatisticalProgramme (NTSYS) software package 1.8 with UPGMA optionand SAHN Clustering (Rohlf 1993). Finally dendrograms show-ing similarity coefficients based on protein or DNA band simi-larity between different plants were prepared.

Results and discussionMorphological markersP. hexandrum populations from Himachal Pradesh exhibitedvariation for number of leaves/plant and leaf shape, etc. Theseresults support the findings of Bhadula et al. (1996) who re-ported morphological variants on the basis of presence of 1, 2, 3


and 4 leaves per plant from the Garhwal region of westernHimalaya. We recorded morphological variation in 200 plants.In general, 37.5% plants had single leaves, 30% had two leaves,22.5% had three leaves and 10% had four leaves. The plantshaving single leaves did not bear fruits. Morphological data for19 plants out of 30 used for protein and DNA analysis arepresented in Table 1.

Polypeptide polymorphismSDS-PAGE of root proteins revealed the presence of 23 bandpositions having proteins with 14 – 100 kDa molecular weight.Out of these 23 markers, seven having molecular weights 77.6,67.6, 48.9, 35.5, 28.8, 23.4 and 15.8 kDa were monomorphic andonly 16 were polymorphic. Since protein profiles revealed alower number of polymorphic markers, the variability generatedusing polypeptide profiles was also low.

Protein data were used to generate a similarity matrix that,after cluster analysis of data, yielded a dendrogram (Fig. 1). At65% level of similarity, the entire population from Chamba andKullu could be divided into two distinct groups (A and B).

These groups, however, were not region specific and also did notcorrelate with leaf number. Group A had 6 plants, 2 fromChamba and 4 from Kullu with Jaccard Coefficient rangingfrom 0.65 to 0.85. Group B was further subdivided into threesubgroups (B1-B3) having genetic similarity values ranging be-tween 78 and 82%. Subgroups B1 and B2 contained plants fromChamba districts with the exception of J13 which was fromKullu and fell into group B2. Subgroup B3 contained 2 plantseach from Chamba and Kullu with genetic similarity of 84%indicating high genetic relatedness in these plants from differ-ent regions. Plants CH44 and J47 of group B were not repre-sented by any of the subgroups B1-B3. Again these subgroupsdid not show any correlation with region specificity or morpho-logical variability in the plants.

Easterase isozyme patterns and polypeptide polymorphismhave already been used to detect genetic variability in P. hexandrumfrom Garhwal Himalaya. However, unlike Purohit et al. (1998)who observed distinct seed polypeptide profiles in differentmorphological variants, we could not observe distinct root pro-tein profiles for different morphological variants.

RAPD markersForty random decamer primers were tested for their ability toproduce DNA polymorphism in genomic DNA of P. hexandrumout of which 33 generated amplification products with a verylow number of bands and/or no polymorphism at all. The datafrom these primers were not included in the final analysis. Theremaining seven primers (Table 2) produced 7 (OPX-1) to 16(OPX-3) amplification fragments with an average of 10.9 bandsper primer. RAPD revealed a high level of DNA polymorphismin P. hexandrum compared with protein profiles. A total of 76RAPD markers were detected from 30 plants with seven primersand all of them showed polymorphism. The size of amplifica-tion products varied from 0.1 to 2 kb.

Cluster analysis of RAPD data based on similarity matrixgenerated a dendrogram (Fig. 2) with six major clusters (I-VI)having similarity coefficient values ranging from 0.25 to 0.75.The morphological variants were not grouped into distinct clus-ters, indicating that genetic differences among them may befew. Populations from Chamba and Kullu regions were clus-tered into region-specific groups with a few exceptions. GroupsI, II, III and VI represented populations from Chamba districtonly. Group I had 3 different plants with Jaccard Coefficientranging from 0.67 to 0.72, group II had 4 plants with Jaccard

Table 1. Podophyllum hexandrum variants showingvariability in leaf number

No. of Plantleaves number

1 CH59, CH44, CH10, CH65, CH70, J35, J132 CH26, CH34, CH30,CH28, CH9, CH513 CH8, CH18, CH11, CH454 CH39, CH61

Fig. 1. Dendrogram of 30 Podophyllum hexandrum plantsgenerated by protein markers. Similarity between plantswas assessed by using Jaccard Coefficient and clusteringwas done by UPGMA. Table 2. List of primers, their sequences and products

generated through amplification

Primer Primer Total Polymorphiccode sequence 5’ – 3’ bands (no.) bands (no.)

OPX-1 CTGGGCACGA 7 7OPX-2 TTCCGCCACC 10 10OPX-3 TGGCGCAGTG 16 16OPX-4 CCGCTACCGA 11 11OPA-1 CAGGCCCTTC 11 11OPA-2 TGCCGAGCTG 10 10OPA-7 GAAACGGGTG 11 11


Coefficient ranging from 0.70 to 0.94, group III had 4 plantswith coefficient of similarity ranging between 0.75 to 0.87, andgroup VI had 3 plants with similarity coefficient of 0.25 to 0.57.Plants from Kullu were grouped into a separate cluster (IV) thatcontained 4 plants. Genetic similarity between populations ofcluster IV ranged from 0.69 to 0.84 with an average of 0.77.Some plants from Chamba and Kullu showed high geneticrelatedness, e.g. group V that contained two plants fromChamba and two from Kullu. Plants from Kullu in group V hadhigh genetic similarity (0.77) compared with Chamba plants.Plants CH30, CH18, CH65, CH61, CH51, CH59, J47 and J18were not represented by any of the groups. The results indicatehigh genetic diversity in P. hexandrum from Himachal Pradesh.

To estimate the potential of individual primers for character-ization of variability in P. hexandrum, data obtained from indi-vidual primers were processed separately (not shown). PrimersOPA-1, OPA-2 and OPX-2 grouped 30 plants into 5 groupseach, OPX-3 into 4 groups and OPA-7, OPX-1 and OPX-4 into 3groups each. However, none of the individual primers couldcluster these plants into region-specific or morphology-specificgroups.

The present study and similar studies on lotus (Campose etal. 1994), sweet potato (Cannoly et al. 1994) and Andrographispaniculata (Padmesh et al. 1999) suggest that RAPD is more appro-priate for analysis of genetic variability in closely related geno-types. The level of genetic variability revealed using RAPD in thepresent study was high compared with protein markers. More-

over, RAPD could differentiate P. hexandrum populations fromChamba and Kullu into distinct region-specific clusters with theexception of J43 and J16 from Kullu indicating that populationsfrom these two regions were genetically distinct. The geneticsimilarity of a few Kullu plants to that of Chamba populationsmight be due to interregional movement of P. hexandrum.

The study also indicates that P. hexandrum populations innorthwestern Himalayan region are genetically highly diverse.The high genetic variation in P. hexandrum may be attributedpartly to the cross-pollinated nature of P. hexandrum. At present,the rate of propagation of P. hexandrum in nature is far less thanthe rate of its exploitation. This species or at least a large part ofits genetic diversity may be lost in the near future owing to itsimportance and consequent exploitation as a medicinal plant, ifappropriate conservation measures are not adopted. Sincesingle or even a few plants will not represent the whole geneticvariability in P. hexandrum, there appears to be a need to maintainsufficiently large populations in natural habitats to conservegenetic diversity in P. hexandrum and avoid genetic erosion. Wehave conserved plants from four locations ex situ at Palampur.However, ex situ conservation was found to cause decline inpodophyllotoxin content in rhizomes and roots of P. hexandrum(Sharma et al. 2000).

ReferencesAiri, S., R.S. Rawal, U. Dhar and A.N. Purohit. 1997. Population

studies on Podophyllum hexandrum Royle – a dwindling me-dicinal plant of the Himalaya. Plant Genet. Resour. Newsl.110:29-34.

Bhadula, S.K., A. Singh, H. Lata, C.P. Kuniyal and A.N. Purohit.1996. Genetic resources of Podphyllum hexandrum Royle, anendangered medicinal species from Garhwal Himalaya, In-dia. Plant Genet. Resour. Newsl. 106:26-29.

Botstein, D., R.L. White, M.H. Skolink and R.W. Davies. 1980.Construction of a genetic map in man using restriction frag-ment length polymorphism. Am. J. Hum. Genet. 32:314-331.

Bradford, M.M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein dye binding. Anal. Biochem. 72:248-254.

Broomhead A.J. and P.M. Dewick. 1990. Tumor inhibitoryaryltralin lignans in Podophyllum versipelle, Diphylleia cymosaand Diphylleia grayi. Phytochemistry 29:3831-3837.

Campose, L.P., J.V. Raelson and W.I. Grant. 1994. Genome rela-tionship among lotus species based on random amplifiedpolymorphic DNA (RAPD). Theor. Appl. Genet. 88:417-422.

Cannoly, A.G., I.D. Godwin, M. Cooper and I.H. Delacy. 1994.Interpretation of randomly amplified polymorphic DNAmarker data for fingerprinting sweet potato (Ipomoea batatasL.). Theor. Appl. Genet. 88:332-336.

Groombridge, B. 1992. Global Bio-diversity Status of the Earth’sLiving Resources, Chapman & Hall, London.

Issell, B.F., F.M. Muggia and S.K. Carter. 1984. Etoposide (VP-16) – Current Status and New Developments. AcademicPress, Orlando, FL, USA.

Jackson, D.E. and P.M. Dewick. 1984. Biosynthesis of Podophyl-lum lignans-II. Interconversion of aryltralin lignan in Podo-phyllum hexandrum. Phytochemistry 23:1037-1042.

Kazan, K., J.M. Manners and D.F. Cameron. 1993. Genetic varia-tion in agronomically important species of Stylosanthes deter-mined using random amplified polymorphic DNA markers.Theor. Appl. Genet. 85:882-888.

Lammeli, V.K. 1970. Cleavage of structural proteins during theassembling of the head of bacteriophage T4. Nature 227:680-685.

Padmesh, P., K.K. Sabu, S. Seeni and P. Pushpangadan. 1999.The use of RAPD in assessing genetic variability inAndrographis paniculata Nees, a hepatoprotective drug. Curr.Sci. 76:833-835.

Fig. 2. Dendrogram of 30 Podophyllum hexandrum plantsgenerated by UPGMA cluster analysis of RAPD data ob-tained with seven 10-mer primers.


Purohit, A.N., H. Lata, S. Nautiyal and M.C. Purohit. 1998. Somecharacteristics of four morphological variants of Podophyllumhexandrum Royle. Plant Genet. Resour. Newsl. 114:51-52.

Purohit, M.C., R. Bahuguna, U.C. Maithani, A.N. Purohit andM.S.M. Rawat. 1999. Variation in podophylloresin and podo-phyllotoxin contents in different populations of Podophyllumhexandrum . Curr. Sci. 77:1078-1080.

Rohlf, F.J. 1993. NTSYS-pc Numerical taxonomy and multivari-ate system, version 1.8. Applied Biostatistics Inc., New York.

Saghai-Maroof, M.A., K.M. Soliman, R.A. Jorgensen and R.W.Allard. 1984. Ribosomal spacer lengths in barley: Mendelianinheritence, chromosomal location and population dynamics.Proc. Natl. Acad. Sci. (USA) 81:8104-8118.

Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Clon-ing. Cold Spring Harbor Laboratory Press, New York.

Sharma, T.R., B.M. Singh, N.R. Sharma and R.S. Chauhan. 2000.Identification of high podophyllotoxin producing biotypes ofPodophyllum hexandrum from north-western Himalaya. J.Plant Biochem. and Biotech. 9:49-51.

Tyler, V.E., L.R. Brady and J.E. Robbers. 1988. Pharmacology. 9 th

edn. Lea and Febiger, Philadelphia.Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V.

Tingay. 1990. DNA polymorphisms amplified by arbitraryprimers are useful as genetic markers. Nucl. Acids Res.18:6531-6535.

62 Plant Genetic Resources Newsletter, 2000, No. 124 Plant Genetic Resources Newsletter, 2000, No. 123: 68 -77

Book ReviewsEncuentro internacional sobre conservación y utilización de recursos fitogenéticos.Valencia, 5-6 de mayo de 1999.Editado por Fernando Nuez Viñals y Juan José Ruiz Martínez. Centro de Conservación y Mejora de la Agrodiversidad, UniversidadPolitécnica de Valencia, España. 1999. ISBN 84-7721-758-0.

La pérdida de la variabilidad genética vegetal es una de lasgrandes preocupaciones del mundo actual y muchosinvestigadores creen que algunas regiones del planeta requierenmás atención que otras. Tal es el caso de la ComunidadValenciana, considerada una de las regiones de Europa quetiene un patrimonio agrícola muy rico y diversificado. LaUniversidad Politécnica de Valencia (UPV) ha pensado en unCentro de Conservación y Mejora de la Agrodiversidad paraevitar la erosión genética y conservar la biodiversidad delpatrimonio hortícola valenciana. La UPV publicó esta obra comoresultado del encuentro de 21 especialistas pertenecientes a 16instituciones y organizaciones de larga trayectoria en el campode los recursos fitogenéticos. Estos investigadores se reunieronpara contribuir a un estudio iniciado por el Banco deGermoplasma de la Universidad Politécnica de Valencia, conmiras a definir los objetivos y la estructura que deberá adoptar elCentro de Conservación y Mejora de la Agrodiversidad.

La obra contiene 8 secciones en que se discuten las ponenciasde los participantes en la reunión. Comprenden ellas la justificaciónde la reunión, las tendencias actuales y las acciones que seestudian para conservar los recursos fitogenéticos, algunosejemplos de modelos reales, los objetivos y la estructura de uncentro de conservación y mejora de la agrodiversidad, lasconclusiones de la reunión y una amplia bibliografía.

l Uno de los principales temas de este documento son lastendencias actuales de los recursos fitogenéticos. Se hace unrecuento histórico que termina en la época actual, para dar allector una idea general sobre el mundo de los recursosfitogenéticos a nivel mundial. Se tienen en cuenta aspectosimportantes que contribuyen a la erosión tanto genética comocultural, y se destaca la importancia del conocimiento humanoen la conservación de los recursos fitogenéticos. Se mencionaluego el sistema mundial de la FAO para la conservación y usosostenible de los recursos fitogenéticos en la alimentación y enla agricultura. Asimismo, se trata el tema relacionado con losderechos de propiedad intelectual, los cuales afectan laconservación y el uso de los recursos fitogenéticos y losderechos de los agricultores.

l Se mencionan también algunas líneas de actuación en elámbito mundial y también en el regional; entre ellas, los programasactuales de la FAO, de UNEP y de UNDP, y la nueva estrategia delIPGRI; las acciones desarrolladas en el ámbito de la UniónEuropea como también en España y en la Comunidad Valenciana.De esta manera se lleva al lector del nivel mundial al local parahacerlo entender la problemática de los recursos fitogenéticos.

Se presentan ejemplos de modelos reales basados enproyectos que se están llevando a cabo en varias regiones en elmundo. Los participantes dan a conocer también ejemplos propiosque han despertado mucho interés por esta obra.

l Otra sección de la obra se dedica a estudiar los objetivosprincipales de un Centro de Conservación y Mejora de laAgrodiversidad. En ellos se contemplan, básicamente, todos lospuntos importantes que comprende el mejoramiento vegetal. Lasvariedades tradicionales suscitan más polémicas, éstas debenmejorarse preservando sus características propias. Se discuteentre los participantes de la reunión sobre quien tiene laresponsabilidad de hacer el trabajo de mejoramiento de lasplantas subutilizadas, deben hacerlo las instituciones públicas olas multinacionales?, pues los intereses son diferentes paraalgunas cultivos.

l Hay, finalmente, una ponencia que presenta la estructuraideal de un centro para conservar y mejorar la agrodiversidad.Se describen aquí las instalaciones y equipamiento que deberíatener dicho Centro. Los participantes hacen recomendacionesbasados en su experiencia y, en algunos casos, describen suslugares de trabajo. Se complementa así el estudio de todos losaspectos importantes y estructuras básicas del Centro.

Esta obra se recomienda a los investigadores y mejoradorespertenecientes a instituciones que trabajan con recursosfitogenéticos. También a los profesionales que deseen enterarsede los aspectos generales del tema. Los editores logran darhábilmente esta visión global a sus lectores.

Dimary Libreros FerlaInformation AssistantIPGRI, Regional Office for the Americas

This book is essential reading for anyone involved in taro geneticresources and taro breeding. It is one of the few attempts that havebeen made to put together a comprehensive treatise on taro, albeitwith a focus on breeding. The review of past work, especially on

the taxonomy and origin of taro, its relationship with other speciesin family Araceae (usually referred to as aroids) and the plant’sreproductive biology is extensive and highly relevant to breedersand scientists involved in taro improvement work. The book draws

The Genetics and Breeding of TaroAnton Ivancic and Vincent Lebot. 2000. Soft cover. CIRAD. ISSN 1251-7224; ISBN 2-87614-414-X


extensively on information gathered in Southeast Asia, the Pacificand Oceania and under the auspices of one of the more successfulnetworks, the Taro Network for Southeast Asia and Oceania(TANSAO), which make it more practical and less theoretical.

As noted by the authors in the foreword, there is frequentconfusion among taro workers between Colocasia esculenta L.Scott and many other aroid species. This book will help correctthis, starting as it does with a well-documented chapter on taroand related aroid species. I am sure that this chapter will be veryuseful for many newcomers as well as old timers. The chapter onsystematics is a bit brief, but necessarily so to meet the objectiveof the book. The problems with respect to origin of genotypes withdifferent ploidy levels are well highlighted; definitely there is aneed for more work to determine their evolution. There seems tobe considerable scope for using techniques such as chloroplastDNA fragment length analysis to clarify some of the problems oftaro origin and taxonomy and domestication issues.

The chapter on genetic resources sums up wells the currentstatus. The authors very carefully point out the need to distinguish‘true’ wild taros from other types of ‘wild’ taros. However, at thistime, it must be noted that the differentiation of wild taro is basedonly on a couple of morphological traits and differences in levels ofoxalates; the higher levels in ‘wild’ taros could be just the result ofnegative selection for this trait under natural conditions. This isanother area that requires use of modern techniques to clearlyidentify true wild material that can actually be used for any desiredtraits it might possess. Reference to conservation through con-serving a random mating panmictic population is probably one ofthe few completely theoretical points that have been made in thisbook. Given the problems of flowering and rapid loss of genes in

succeeding generations, this is far from reality.The status of conservation is well reviewed. This is a very

dynamic area, as many accessions are lost in field genebanksdue to vagaries of environment at each re-planting. Most of thecharacterization and evaluation work that is described is currentand some of the results will be fully available in a year or two. Thesection on global genetic resources is important, as it relates tosome of the questions that FAO and IPGRI are asking aboutseveral crop species, including taro. It is, however, brief anddoes not deal with questions such as uniqueness of the collec-tions, quality of conservation efforts, including documentation,status of characterization and evaluation and regeneration ofgermplasm and access to the germplasm.

The chapter on reproductive biology is well researched and isextremely useful, in the contexts of both taro breeding and con-servation through true seed. The chapter on breeding, probablycentral to the book, is written in the manner of very practicalguide. Again this chapter is based on long personal experience ofauthors and even small details are well illustrated. Authors alsopoint out, in very practical terms, things that need to be doneaccording to the aims of the breeding programme, for exampledisease resistance or density resistance (the later I would preferto refer as suitability to close planting, though).

As authors point out, it should be possible to make taro breed-ing simpler, more efficient and more pleasant by sharing much ofthe valuable information that stems from the authors’ experienceswith this interesting, but under-researched and underutilized crop,taro. This book goes a long way towards achieving this aim.

Ramanatha Rao

2000 RefereesThe Plant Genetic Resources Newsletter peer reviews all of the reviews, articles and short communication it receives. Since thisprocess is confidential, it is a pleasure to acknowledge here the scientists from around the world who have selflessly given up theirtime to act as referees and maintain the standard of the Newsletter.

Ambrose MikeArora R.K.Bellon MauricioBorelli SimoneCoppens GeoDebouck DanielEngelmann FlorentEskes BertusFernando UrslaFrese LotharFundora Mayor ZoilaGrum MikkelGuarino LuigiHamon SergeHodgkin TobyHoogendijk MichelHulden MortenLastra Ramon

Leal FreddyLester R.N.Maggioni LorenzoMaxted NigelMeilleur Brien A.Metz ThomasMorales FranciscoPadulosi StefanoRao RamanathaRocha O.J.Ruiz MagdalenaTripp Robertvan Hintum Theovan Soest J.M.von Bothmervon Mark CruzWatanabe K.Williams David

Plant Genetic ResourcesNewsletter

Aims and scopeThe Plant Genetic Resources Newsletter pub-lishes papers in English, French or Spanish,dealing with the genetic resources of useful plants,resulting from new work, historical study, reviewand criticism in genetic diversity, ethnobotanicaland ecogeographical surveying, herbarium stud-ies, collecting, characterization and evaluation,documentation, conservation, and genebank prac-tice.

ManagementThe Plant Genetic Resources Newsletter is pub-lished under the joint auspices of the Internation-al Plant Genetic Resources Institute (IPGRI) andthe Plant Production and Protection Division ofthe Food and Agriculture Organization of theUnited Nations (FAO).

AvailabilityThe Plant Genetic Resources Newsletter ap-pears as one volume per year, made up of fourissues, published in March, June, September andDecember. Plant Genetic Resources Newsletteris available free of charge to interested librariesof genebanks, university and government depart-ments, research institutions, etc. The periodicalmay also be made available to individuals whocan show that they have a need for a personalcopy of the publication.

Types of paperArt ic lesAn article will publish the results of new andoriginal work that makes a significant contribu-tion to the knowledge of the subject area that thearticle deals with. Articles, which should be of areasonable length, will be considered by the Edi-torial Committee for scope and suitability, thenassessed by an expert referee for scientific con-tent and validity.

Short communicationsA short communication will report results, in anabbreviated form, of work of interest to the plantgenetic resources community. Short communi-cations in particular will contain accounts of ger-mplasm acquisition missions. The papers will beassessed by an expert referee for scientific con-tent and validity.

Other papersThe Plant Genetic Resources Newsletter willpublish other forms of reports such as discussionpapers, critical reviews, and papers discussingcurrent issues within plant genetic resources.Book reviews will be printed, as well as a Newsand Notes section. Suggestions for books toreview are invited, as are contributions to Newsand Notes.

SubmissionIn the first instance papers may be submitted intypescript form or as an Email message. Thefinal version may be submitted as an Email file oras a Windows-readable file on diskette. Manu-scripts submitted for publication and other com-munications on editorial matters should be ad-dressed to IPGRI's Editorial and PublicationsUnit.

Bulletin des ressourcesphytogénétiques

Domaine d’intérêtLe Bulletin des ressources phytogénétiques pub-lie des articles en anglais, en espagnol et enfrançais, sur les ressources génétiques de plan-tes utiles, fruit de nouvelles recherches, d’étudeshistoriques, d’examens et de critiques concer-nant la diversité génétique, d’études ethnobota-niques et écogéographiques, d’études d’herbiers,d’activités de collecte, de caractérisation etd’évaluation, de documentation, de conservationet les pratiques des banques de gènes.

ParrainageLe Bulletin des ressources phytogénétiques estpublié sous les auspices de l’Institut internationaldes ressources phytogénétiques (IPGRI) et de laDivision de la production végétale et de la protec-tion des plantes de l’Organisation des NationsUnies pour l’alimentation et l’agriculture (FAO)

DistributionLe Bulletin des ressources phytogénétiques paraîtune fois par an en un volume regroupant quatrenuméros publiés en mars, juin, septembre etdécembre. Il est distribué gratuitement aux bib-liothèques des banques de gènes, universités,services gouvernementaux, instituts de recher-che, etc. s’intéressant aux ressources phytogéné-tiques. Il est aussi envoyé sur demande à tousceux pouvant démontrer qu’ils ont besoin d’unexemplaire personnel de cette publication.

Types de documents publiésArt ic lesUn article contient les résultats de travaux nou-veaux et originaux qui apportent une contributionimportante à la connaissance du sujet dont traitel’article. Les articles, qui doivent être d’unelongueur raisonnable, sont d’abord examinés parle Comité de rédaction qui en évalue la portée etla validité, puis par un expert qui en examine lecontenu et l’intérêt scientifiques.

Brèves communicationsOn entend par brève communication un textecontenant, sous une forme abrégée, les résultatsde travaux présentant un intêrêt pour tous ceuxqui s’occupent de ressources phytogénétiques.Elle contient en particulier des comptes rendusdes missions d’acquisition de matériel génétique.

Autres documentsLe Bulletin des ressources phytogénétiques pub-lie d’autres types de rapport tels que des docu-ments de synthèse, des études critiques et desarticles commentant des problèmes actuels con-cernant les ressources phytogénétiques. Le Bul-letin publie une revue de livres ainsi qu’une sec-tion intitulée Nouvelles et Notes. Les auteurssont invités à envoyer leurs suggestions pour leslivres à passer en revue ainsi que des contribu-tions aux Nouvelles et Notes.

PrésentationEn premier lieu, les documents doivent être sou-mis dactylographiés ou par courrier électronique.La version définitive doit être présentée en fichierde courrier électronique ou sur disquettes com-patibles Windows. Prière d’adresser les manuscritsprésentés pour être publiés et d’autres communi-cations sur des questions de rédaction au Bureaude rédaction de l'IPGRI.

Boletín de RecursosFitogenéticos

Objetivos y temasEl Noticiario de Recursos Fitogenéticos publicadocumentos en inglés, francés y español quetratan de los recursos genéticos de plantas útiles,fruto de nuevos trabajos, estudios históricos,revisiones y análisis críticos relacionados con ladiversidad genética, investigaciones etnobotáni-cas y ecogeográficas, estudios de herbarios,actividades de colección, caracterización y eval-uación, documentación, conservación, y prácti-cas en bancos de germoplasma.

DirecciónEl Noticiario de Recursos Fitogenéticos se publi-ca bajo los auspicios conjuntos del Instituto In-ternacional de Recursos Fitogenéticos y la Di-rección de Producción y Protección Vegetal de laOrganización de las Naciones Unidas para laAgricultura y la Alimentación.

DistribuciónEl Noticiario de Recursos Fitogenéticos aparececomo un volumen anual compuesto por cuatronúmeros, que se publican en marzo, junio, septi-embre y diciembre. Se distribuye gratuitamente alas bibliotecas de bancos de germoplasma, facul-tades universitarias y servicios gubernamentales,centros de investigación, etc. que se interesanen los recursos fitogenéticos. También puedenobtener este noticiario las personas que demues-tren necesitar una copia personal.

Tipos de documentosArtículosLos artículos divulgarán los resultados de traba-jos nuevos y originales que contribuyan de modoimportante al conocimiento del tema tratado.Dichos artículos, que deberán tener una longitudrazonable, serán examinados por el Comité deRedacción en cuanto a su pertinencia e idoneidady posteriormente un experto juzgará su contenidoy validez científicos.

Comunicaciones brevesLas comunicaciones breves informarán de modoconciso sobre los resultados de trabajos de in-terés para las personas que se ocupan de losrecursos fitogenéticos. Las comunicacionesbreves incluirán, en particular, resúmenes sobrelas misiones de adquisición de germoplasma.

Otros documentosEl Noticiario de Recursos Fitogenéticos publi-cará otros tipos de informes, como documentosde trabajo, análisis críticos, y documentos queexaminen cuestiones de actualidad relacionadascon los recursos fitogenéticos. El Noticiario pub-licará una reseña de libros así como una secciónde Noticias y Notas. Las propuestas de librospara reseñar y las contribuciones a la sección deNoticias y Notas serán bien acogidas.

PresentaciónLos documentos deben entregarse, incialmente,en forma de texto mecanografiado o a través delcorreo electrónico. La versión final debe presen-tarse como un archivo de correo electrónico o endisquete compatible con el sistema operativoWindows. Los manuscritos para publicar y otrascomunicaciones sobre asuntos relativos a la re-dacción deberán dirigirse a la Oficina de Redac-ción del IPGRI.

No. 124, December 2000

Contents


Articles

Agronomic assessment of wild cocoa trees (Theobroma cacao L.) from the Camopi andTanpok basins (French Guiana)Ph. Lachenaud (Ivory Coast), G. Oliver and Ph. Letourmy (France) .................................................................................... 1

Interacciones genéticas entre germoplasma silvestre y cultivado de Lycopersicon spp.con efectos sobre la calidad del fruto de tomateG. Pratta, R. Zorzoli y L.A. Picardi (Argentina) ............................................................................................................................ 7

Cultivation and use of African yam bean (Sphenostylis stenocarpa) in the Volta Region of GhanaG.Y.P. Klu, H.M. Amoatey, D. Bansa and F.K. Kumaga (Ghana) ...................................................................................... 1 3

Location of an endophytic Neotyphodium sp. within various leaf tissues of wild barley(Hordeum brevisubulatum subsp. violaceum)N.N. Youssef and F.M. Dugan (USA) ............................................................................................................................................. 1 7

Evaluation and characterisation of sugar cane germplasm accessions for their breeding values in NigeriaS. Agboire, A.C. Wada and M.N. Ishaq (Nigeria) ....................................................................................................................... 2 0

The significance of Vavilov's scientific expeditions and ideas for development anduse of legume genetic resourcesB.S. Kurlovich (Finland), S.I. Rep'ev, M.V. Petrova, T.V. Buravtseva, L.T. Kartuzova andT.A. Voluzneva (Russia) ..................................................................................................................................................................... 2 3

Impact of cultivation on active constituents of the medicinal plants Podophyllum hexandrum andAconitum heterophyllum in SikkimP. Prasad (India) .................................................................................................................................................................................... 3 3

Morpho-agronomic variability of the diploid wheat Triticum monococcum L.S. Empilli, R. Castagna and A. Brandolini (Italy) ....................................................................................................................... 3 6

Polyphenol oxidase activity as an index for screening mango (Mangifera indica L.) germplasmagainst malformationR.R. Sharma, C.N. Singh, O.P. Chhonkar, A.M. Goswami and S.K. Singh (India) ...................................................... 4 1

A nested analysis to detect relationships between genetic markers and germplasm classesof durum wheatG. Figliuolo and P.L. Spagnoletti Zeuli (Italy) ............................................................................................................................. 4 4

Short Communications

Use of conserved rice germplasmG.C. Loresto, E. Guevarra and M.T. Jackson (Philippines)........................................................................ 51

Molecular analysis of variability in Podophyllum hexandrum Royle - an endangered medicinal herbof northwestern HimalayaK.D. Sharma, B.M. Singh, T.R. Sharma, M. Katoch and S. Guleria (India)................................................... 57

Book Reviews ........................................................................................................................................................................................ 6 22000 Referees ......................................................................................................................................................................................... 6 3

Documents

Plant Genetic Resources newsletter No. 124, December 2000