Euphytica 94: 367–373, 1997. 367c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Genetic potential and stability of carotene content in cassava roots �

Carlos Iglesias1;�, Jorge Mayer1, Lucıa Chavez1 & Fernando Calle1

1Cassava Program and Biotechnology Research Unit of CIAT, A.A. 6713, Cali, Colombia; (�author forcorrespondence)

Received 4 June 1996; accepted 31 December 1996

Key words: cassava, Manihot esculenta, vitamin A, carotene

Summary

People in vast areas of the tropics suffer from vitamin A deficiency, resulting in progressive eye damage andeventually leading to blindness. Improving the content of vitamin A precursors in staple crops could alleviate orsolve such a problem. The objective of this work was to study the range of variability for carotene content in asub-set of the global cassava germplasm collection, and to determine the inheritance of carotenes, as well as theirstability in response to different processing methodologies. Genotypes with more than 2 mg carotenes/100 g offresh roots have been selected as parental material for population development. Although root colour is highlycorrelated with carotene content, a quantitative evaluation of genotypes selected by colour is required in orderto increase the efficiency of selection. Relatively few major genes are involved in the determination of caroteneaccumulation in cassava roots. Stability of carotenes in response to different processing methods is genotypicallydependant, representing a trait to be evaluated after selecting for high carotene concentration in fresh roots. Theresults from this work have provided the basis for defining future strategies for the improvement of the nutritionalquality of cassava.

Introduction

Cassava is one of the most important sources of foodenergy in several tropical countries. There are an esti-mated 70 million people who obtain more than 500cal/day from cassava, particularly in Africa and NEBrazil (Cock, 1985). Cassava cab produce resonablywell under marginal conditions of climate and soil. Thecrop is frequently identified as a famine reserve due toits tolerance of drought and infertile soils, and its abil-ity to recover from disease and pest attacks. Cassavaalso offers the advantage of a flexible harvesting date,allowing for farmers to keep the roots in the grounduntil needed. The area of cassava under marginal envi-ronments has been continuously increasing, particular-ly for regions with poorer soils and lengthy dry seasons(El-Sharkawy, 1994). As a consequence, any improve-ment of the efficiency with which cassava accumulatesmicro-nutrients and vitamins in the roots and leaves,could have great potential not only in terms of human

� Research supported in part by grant C-94050 of IFPRI.

nutrition, but also in relation to the crops ability tocope with unfavorable environments. Some of thosemicro-nutrients and vitamins play a central role in theplant metabolism, and enhancing their acquisition canhave a direct positive effect in crop production.

Vitamin A deficiency, defined as eye signs, has beenidentified as a widespread public health problem in 37countries worldwide, affecting a considerable percent-age of the population in regions where cassava is a sta-ple (NE Brazil; Sub-sahelian Africa; and S-SE Asia),(Schrimpton, 1989; WHO, 1995). A valid strategy toapproach this problem is to enhance the nutritional val-ue of cassava through plant breeding. The compositionof cassava roots and leaves depends both on the geneticpotential of varieties and on the edapho-climatic con-ditions under which they have been grown (Howelerand Cadavid, 1983).

The average daily requirement of�-carotene equiv-alent for children is 2.4 mg; that of adults is 3.5 mg, andthat of lactating women is 5.0 mg (WHO, 1995). Thecontinued consumption of vitamin A deficient diets

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results in xerophthalmia, which can range from mildforms of night blindness to ulceration and destruc-tion of the cornea (WHO, 1995). Alternative improvedforms of existing staple crops should be able to at leastcover the daily requirements of vitamin A, within therange of daily consumed product. Through the processof selection and recombination, the levels of the mostimportant nutrient components can be improved toreach significant levels in human nutrition.

At the beginning of any breeding program, it isimportant to evaluate the available genetic variability.The potential of the species as a source of the micro-nutrient under consideration can thus be determined,and an ideas of the maximum levels attainable throughselection and recombination can be obtained. Reportson variability for carotene content among accessions ofnational cassava germplasm collections have been pub-lished for India (Moorthy et al.; 1990) and for Brazil(Ortega-Flores; 1991). The ranges reported by theseauthors did not exceed 0.8 mg of �-carotene equivalentper 100 g of fresh roots. On the other hand, Jos et al.(1990) demonstrated the potential for rapidly increas-ing carotene content in cassava roots through cycles ofrecurrent selection. They were able to increase the con-centration from 0.42 mg/100g of fresh roots in the basepopulation, to 1.38 mg/100g after 2 cycles of selectionand recombination.

The relationship between yellow color and concen-tration of pro-vitamin A compounds in cassava rootshas long been established. Meanwhile, the color ofroot parenchyma seems to be simply inherited. It wasreported by Hershey and Ocampo (1989) that a singledominant factor was responsible for the yellow color,that recessive alleles determined white color and thatthe heterozygous was cream. If the genetics of pro-vitamin A concentration follow that pattern, it will berelatively easy to manipulate within segregating proge-nies.

Nutritional value is not so much defined by theconcentration of a nutrient in the crop itself, but ratherby the concentration which remains once the crop hasbeen processed prior to its consumption. Cassava rootsare usually processed before consumption, either byboiling, roasting or drying (Cock, 1985). It is expectedthat not all of the pro-vitamin A present in the rootswill be available after processing. The stability of pro-vitamin A compounds might be genotype-dependent.

The objectives of this work, the first screening of asub-set of the global cassava germplasm for carotenecontent, are: a) to determine the range of caroteneconcentration by screening roots of cassava landraces

from CIAT’s germplasm collection; b) to correlatecarotene content with root color; c) to study the genet-ics of carotene accumulation in cassava roots; and d) todetermine carotene losses and degradation in processedproducts.

Materials and methods

Pigment extraction and evaluation procedure

An extraction procedure was adjusted for cassava rootparenchyma, taking as a base the one outlined by Safo-Katanga et al. (1984). Root parenchyma was extract-ed with petroleum ether. After extraction, the organicphase is dried with sodium sulfate and concentrated byrotatory evaporation, before injecting into the HPLCcolumn. HPLC analysis was performed using a Hyper-sil ODS column (250 x 4 mm) with 5 �m particlesize. A chloroform-methanol gradient (0-100% in 20minutes, flow rate of 1 ml/min), was used, giving aretention time of 9.2 minutes and good peak separa-tion (better than isocratic chloroform and actonitril-chloroform gradients). UV detection was at 455 nm.A calibration curve was determined (1 to 20 �g/gcarotene). Peaks were identified on the basis of co-migration and shared spectro-photometric profiles withknown standards.

Plant material

Plants were sampled between 10 and 11 months afterplanting. A sample of 10 g was taken out of the centralpart of one root, taken at random from a plant in theaccession plot.

A sub-set of 632 accessions from the cassavagermplasm collection was evaluated, without repli-cation, for carotene content in the roots to study therange of genetic variability. Thirty-nine individualsfrom a cross between a yellow-root (CM 2772-3) anda white-root genotype (CG 1372-6) were sampled (2roots/plant, average data used for the analysis) togetherwith the parental material for the inheritance study.

The work on carotene stability in response to differ-ent processing methods was conducted on 28 yellow-root genotypes from the germplasm collection, grownwith 2 replications. The treatments were: a) fresh,unprocessed root parenchyma as a control (FR); b)root parenchyma cooked for up to 30 minutes (CR);c) flour obtained by oven drying cassava chips and

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milling (CFO); and d) flour obtained by sun dryingcassava chips and milling (CFS).

Statistical analysis

Simple correlation analysis was conducted to establishthe relationship between root colour and carotene con-tent, and among different processing methods. A ChiSquare test was run to determine the goodness of fitbetween observed and predicted values for segregatingprogenies. An ANOVA and treatment mean compari-son by LSD (5%) were carried out.

Results and discussion

Screening of cassava landraces from CIAT’sgermplasm collection

A total of 632 accessions from the CIAT globalgermplasm collection of 5500 accessions, selectedaccording to parenchyma colour were screened forcarotene content. Although the previous colorimetricscale defined for cassava germplasm characterizationin terms of root parenchymal colour (white, creamand yellow) (Hershey, 1987) was very useful, thiswork showed considerable variation with respect tothe intensity of yellow, some root parenchyma beingcloser to orange.

The quantitative data showed a broad distribu-tion of concentrations from less than 0.1 to 2.4 mgcarotenes/100 g of fresh roots. The relatively sim-ple composition of cassava roots (not many com-pounds interfering with carotene extraction) enabledthe simplification of the normal procedure by extract-ing directly with petroleum ether (30/60), instead ofdi-cloro methane; thereby lowering the cost per extrac-tion from U$ 4.33 to U$ 2.85 per sample. The resultsare expressed in fresh rather than dry basis in orderto have a direct idea of the nutritional potential ofeach accessions when consumed fresh. Although thereis a close relationship between the quantitative lev-els of carotene and the colour of root parenchyma,the variability observed among genotypes with simi-lar parenchymal color resulted in an overlap betweenwhite and cream roots, and between cream and yellowroots. Table 1 presents the mean values and standarddeviations for groups of accessions classified accordingto root parenchymal colour. Even those deep-yellowand orange roots showed a broad range of concentra-tions, from 0.6 to 2.4 mg carotenes/100 g fresh root.

Table 1. Average carotene concentration in cassava rootsclassified according to root colour

Root color Numerical Carotene Standard

scale (mg/100g) deviation

White 1 0.13 0.48

Cream 2 0.39 0.28

Yellow 3 0.58 0.28

Deep yellow 4 0.85 0.17

Orange 5 1.26 0.11

Although it seems feasible to improve �- carotene lev-els through visual selection for root colour, there is aneed for quantitative screening in order to increase theefficiency of the process. Other pro-vitamin A com-pounds should also be quantified, because they maybe responsible for deep yellow color in accessions thathave intermediate carotene concentrations.

It seems possible to select genotypes with 2 mgof carotene/100 g of root out of the available geneticvariability (Table 2), which sets a considerably high-er upper limit than previous reports (Jos et al., 1990;Moorthy et al., 1990). Given that the average dailyrequirements of vitamin A is around 3 mg (WHO,1995); the consumption of 150 g of roots from geno-types with such high concentration might supply thisrequirement, provided that the carotene is 100% avail-able to the human organism.

The 5 genotypes having the highest�-carotene con-centration were collected in the Amazonian region ofBrazil and Colombia, where yellow parenchyma culti-vars are preferred by farmers (Dufour, 1993) (Table 2).This group of top accessions for carotene concentra-tion represents the basis for the future recombinationwork. It is expected that transgressive segregants couldbe selected out of the recombinant progenies. Anotherimportant aspect is the fact that selection for caroteneconcentration does not necessarily mean selection foragronomic traits, production potential and other rootquality traits. Building a genetic stock for high carotenewith sufficient genetic variability might permit selec-tion for other important characteristics.

In order to estimate the correlation between rootparenchyma color and �-carotene content, a transfor-mation of the qualitative data for root color was nec-essary. In the evaluation scale for germplasm charac-terization, root parenchyma colour has been classifiedas: 1=white; 2=cream and 3=yellow (Hershey, 1987),with each increment in color intensity representing aproportional increment in carotene concentration. Tak-

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Table 2. Cassava genotypes with the highest concentration of carotene (fresh weight basis) in theroots

Genotype Common Name Origin Root color Carotene

(mg/100 g)

MBRA 516 Olho Verde Manaus - Brazil Deep yellow 2.55

MCOL 2285 Furetsikae Guainia - Col. Orange 2.40

MCOL 2109 Manaca Amazonas - Col. Orange 2.14

MCOL 2295 Bitsurikae Guainia - Col. Orange 2.12

MBRA 481 Baixota Manaus - Brazil Orange 2.04

ing into account the results from the carotene evalua-tion among germplasm accessions (Table 1), a broaderrange of root colour and related carotene content wasrevealed. Data also showed that the color "cream" isnot exactly half-way between white and yellow, but ittends towards yellow, meaning that the yellow colourhas a degree of dominance. This work also revealedgenotypes with higher colour intensity than yellowwithin the range of available variability. Therefore,a more appropriate numerical scale for root colour wasdefined: 1=white; 2=cream; 3=yellow; 4= deep yellow;5=orange.

From the analysis of the 632 accessions, a sig-nificant correlation (r=0.82) between root color andcarotene content, was determined. It means that 67%of the total variability in carotene content can beexplained by the variability in root color. If pooled datafor carotene content within a particular colour class istaken for the correlation analysis (Table 1), then 98%of the variability in carotene content can be explainedby the variability in root colour. Therefore, in general itis possible to improve carotene content by visual selec-tion for color intensity. Among genotypes classified inthe upper colour class there is scope to improve selec-tion efficiency by quantitative evaluation of carotenecontent, as a result of the observed variability amongsamples.

Inheritance of carotene content in cassava roots

The biochemical pathway leading to the synthesisof carotenes has been long established (Spurgeon &Porter, 1980). Single breaks in the pathway may resultin lack or reduced levels of carotene formation. A pre-vious report by Hershey and Ocampo (1989) estab-lished that root color (white/cream/yellow) was deter-mined by a partially dominant gene. Although, that canbe true for parents differing in allele composition forone locus, recently observed segregations, have sug-

Table 3. Segregation for root colour and carotene concentra-tion (fresh weight basis) in a cross between contrasting parents

Genotypes Number of Number of Carotene

individuals individuals (mg/100 g)

(Observed) (Expected)

CM 2772-3 (yellow) 0.42

CG 1372-6 (white) 0.08

White 20 19.50 0.09

Cream 10 9.75 0.28

Yellow 9 9.75 0.38

gested that the inheritance of root colour, and thereforecarotene content, was more complex.

During the previous growing cycle and on singleplant basis, the hypothesis of two genes with epistat-ic effects controlling root colour, was developed froma cross between a white-root and a yellow-root geno-type. These genes were nominated as Y1 with completedominance, allowing for the transport of carotene athigh levels to the roots; and Y2 with partial dominanceallowing for the accumulation of carotene in the roots.The genotype of the white-root parent was assumed tobe y1y1Y2y2, and the yellow-root parent as Y1y1Y2Y2,according to the observed segregation in the F1. Theexpected segregation from those genotypes was 50%white (y1y1Y2-), 25% cream (Y1y1Y2y2), and 25% yel-low (Y1y1Y2Y2). According to the Chi square test, theobserved segregation has a probability between 80 and90% of supporting the original hypothesis. The par-ent/progeny performance for root color and carotenecontent is presented in Table 3. Our study included twocontrasting parents, rated 1 and 3 in the new scale. Thissame study conducted with parental material present-ing more extreme root colors (i.e. white and orange)can reveal more complex inheritance, as more alle-les might get involved in the genetic determination ofcarotene content.

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Table 4. Analysis of variance for the experiment involvingdifferent root processing treatments and their effects oncarotene content

Source of Degrees of Sums of Mean F values

variation freedom squares squares

Reps 1 0.05 0.05 0.1

Treatments 111 195.87 1.76 3.9��

Varieties 27 85.88 3.18 7.0��

Processing 3 80.56 26.85 59.2��

V x P 81 29.43 0.36 0.8

Error 111 12.24 0.45

��: significant at 1% probability.

Table 5. Correlation (r) among processing methodolo-gies for carotene concentration within a group of 28yellow-root genotypes [control (FR), cooked parenchy-ma (CR), oven dried flour (CFO) and sun dried flour(CFS)]

FR CR CFO

CR 0.80��

CFO 0.62� 0.70��

CFS 0.59� 0.76�� 0.79��

�, ��: significant at 5% and 1% probability, respectively.

A careful planning of crosses and progeny testingis needed in order to recover high carotene genotypes.Although major genes dominate the transport and accu-mulation of carotene in the roots, the quantitative vari-ability observed within root color classes suggestedthat a number of genes with smaller effects are involvedin the accumulation process. Therefore, there is goodscope to achieve maximum levels of expression by aprocess of recurrent selection (Jos et al., 1990).

Stability of carotenes after processing cassava roots

Cassava is processed before consumption. It is eitherboiled or transformed to different kinds of flours orstarches. During processing, the product is exposed tosome heat treatment, which can affect carotene concen-tration and availability. The effects of different process-ing methodologies [FR (control), CR, CFO, and CFS,as described in Materials and Methods] were studiedin a group of 28 genotypes.

The ANOVA (Table 4) showed a very large and sig-nificant effect of processing, followed by the effect ofvarieties. Data were transformed to concentration ondry basis in order to make it comparable across process-ing methods. On average, simple boiling reduced

Table 6. Effect of root processing on the concentrationof carotene in different cassava genotypes [control (FR),cooked parenchyma (CR), oven dried flour (CFO) and sundried flour (CFS)]

Carotene (mg/100 g dry root)

Genotype FR (control) CR CFO CFS

MBRA 478 4.69 3.03 1.72 0.77

MBRA 476 4.50 1.68 1.60 0.56

MBRA 473 3.33 3.25 2.10 1.15

MBRA 466 3.21 3.12 1.01 0.76

MBRA 502 3.14 1.67 0.69 0.66

MBRA 437 2.87 2.37 1.52 0.49

MBRA 928 2.66 1.07 0.44 0.27

MBRA 489 2.37 0.78 1.03 0.64

MBRA 467 2.19 2.15 1.81 1.19

CM 8371-27 2.14 0.92 0.77 0.25

MBRA 518 2.07 0.77 0.47 0.49

MBRA 463 1.91 1.75 1.15 0.52

MBRA 458 1.76 1.00 0.56 0.24

MBRA 734 1.67 1.57 0.46 0.33

CM 8371-25 1.55 0.73 1.30 0.33

CM 8371-32 1.44 0.67 1.13 0.45

CM 8371-38 1.23 0.62 0.61 0.37

MCOL 2290 1.19 0.40 0.28 0.24

CM 8371-11 1.14 0.45 0.62 0.14

CM 8371-40 1.10 0.45 1.06 0.48

CM 8371-35 1.06 0.65 0.79 0.41

CM 8371-31 0 93 0.73 0.87 0.51

CM 8371-24 0 93 0.55 0.82 0.33

CM 8371- 3 0.89 0.70 0.36 0.36

CM 8371-10 0.87 0.83 0.86 0.47

CM 8371- 5 0.86 0.37 0.60 0.21

CM 8371- 7 0.84 0.70 0.41 0.13

CM 8371-16 0.77 0.43 0.53 0.10

Trial mean 1.89 1.20 0.92 0.46

carotene content the least (34% reduction), followedby oven dried flour (CFO) with a 44% reduction. Sundried flour (CFS) reduced the carotene concentrationto the lowest level (73% reduction), which seems toimply that carotenes are photo-labile. Although thecorrelation among different processing methods acrossgenotypes was significant (Table 5 and 6), the relativemagnitude of the effects (r2), indicates that the geno-types with the highest carotene concentration in thefresh root controls are not the ones with the highestconcentration after processing. Therefore, after rou-tine screening for high �-carotene in fresh roots, a testof stability after different processing methods shouldalso be routinely carried out.

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Table 6 presents the complete data set for the geno-type by processing method interaction. The carotenecontent of genotype MBRA 473 was found to be themost stable. In spite of not having one of the highestconcentrations of carotene in FR, it showed the highestcontent for CR and CFO treatments, and the secondhighest for CFS treatment. Genotypes MBRA 502 andMBRA 476, with the same or higher concentrations inFR, showed a reduction to more than half the originalcontent, after the different processing.

In spite of the processing steps involved in pro-ducing dry cassava flour, considerable amounts ofcarotenes were conserved, particularly when oven dry-ing was carried out. These results are supportive ofthe findings of McDowell and Oduro (1981). Gari(a dry flour produced from cassava roots) obtainedfrom yellow-root cultivars, presented concentrationsof carotene of up to 1.13 mg/100 g. In relation tothe nutritional value of processed cassava foods, it isalso important to study how much carotene left afterprocessing is available to the human body, once thecassava product is consumed.

Summary and conclusions

A set of trials were carried out to study variabilityfor �-carotene content in a sub-set of CIAT’s cassavagermplasm collection, its relationship with root col-or, its heritability, and its stability after different rootprocessing procedures.

Within the range of variability for carotene con-centration in the roots, a few genotypes with more than2 mg/100 g of roots may be selected. These high lev-els should be combined with other agronomic and rootquality traits in order to have cassava which is as goodin production and quality as the most popular cultivarsin the different regions, but with the added benefit ofhigh carotene content.

Intensity of root color is correlated with caroteneconcentration, but the deviations do justify a quantita-tive evaluation of carotene of genotypes pre-selectedaccording to root parenchyma color. The inheritanceof carotene concentration might be determined by twogenes, one controlling the transport of the product ofprecursors to the roots, the other responsible for theaccumulation process. The genes have epistatic effects.Other major genes not segregating in the cross that wasstudied, will have to be studied in the future, as well asgenes with minor effects.

High levels of carotene can be recovered after boil-ing or the production of cassava flour artificially dried,this recovery varied depending on the genotype underevaluation. Therefore, after selecting genotypes withthe highest concentration of carotene in fresh roots, anevaluation of processing effect, should be carried out.

As soon as genotypes with high concentration andhigh recovery rates are available, the bio-availabilityof the carotenes should be evaluated, in order to deter-mine the potential of cassava roots as a food sourceof pro-vitamin A compounds. Selected genotypes rep-resent the basis for a recurrent selection programmeto increase the levels of carotene in cassava roots, tolevels beyond the ones actually found in the cassavagermplasm collection. Carotenes are associated withthe photosynthetic process, serving as anti-oxidants(Bartley et al., 1994). Cassava leaves could also repre-sent an important food source of vitamins and minerals(Bokanga, 1994). Therefore, a study of the concentra-tion of carotene in cassava leaves of those selectedgenotypes should be carried out, as well as its stabilityafter processing. Due to the high levels of cyanogenicglycosides in the leaves, even more elaborate process-ing may be required to make them amenable forhuman consumption, whilst assuring conservation ofcarotenoid compounds.

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