Proc. Natl. Acad. Sci. USAVol. 91, pp. 12877-12881, December 1994Agricultural Sciences

High a-tomatine content in ripe fruit of Andean Lycopersiconesculentum var. cerasiforme: Developmental and genetic aspectsCHARLES M. RICK*t, JOHN W. UHLIGt, AND A. DANIEL JONES§*Department of Vegetable Crops, and §Facility for Advanced Instrumentation, University of California, Davis, CA 95616; and iPetoseeds Research, 37437State Highway 16, Woodland, CA 95695

Contributed by Charles M. Rick, August 8, 1994

ABSTRACT A variant of Lycopersion esculentum var.cerasiforme is described that deviates from the typical form ofthe entire species, including cultivated tomatoes, in possessinghigh levels (500-5000 lAg/g of dry weight) of the steroidalalkaloid a-tomatine in its ripe fruits. This biotype is restrictedto a tiny enclave in the valley of Rio Mayo, Department SanMartin, Peru. Among 88 accessions of var. cerasiforme from itspresent distribution in the Andes, a 90% association was foundbetween high tomatine and bitter flavor; within the Mayowatershed, all samples from the upper drainage had bitternessand high tomatine; the frequency ofboth traits decreased to lowlevels toward the lower end. Tomatine therefore probably is thesource of bitterness. Throughout L. esculentum tomatine ispresent at very high concentrations in earliest stages of fruitdevelopment, thereafter decreasing rapidly to midperiod, andfinally diminishing gradually to near zero at maturity as aresult of catabolism to biologically inert compounds, except inthe variant described here. High tomatine content does notappear to affect adversely either the natives, among whom thebitter types are popular, or individuals who sampled them inthis survey. Genetic determination of high tomatine in ripefruits is totally recessive and appears to be monogenic withinteraction with genes of minor effect. The prevailing patternof glycoalkaloid synthesis and degradation in development ofsolanaceous fruits suggests a mechanism to protect againstpredation prior to ripening but to permit it afterward as adevice to promote dispersal. In consideration of the nondeg-radative nature of the variant, its genetic determination, andvery restricted geographic distribution, mutation to this formappears to be a random event of doubtful evolutionary signif-icance.

In a survey of genetic variation in wild cherry tomatoes(Lycopersicon esculentum var. cerasiforme, sensu latu) ofthe northwest Andes, variation was encountered in bitternessof ripe fruit in the valley of Rfo Mayo, Department SanMartin, Peru (1). Bitter accessions were concentrated in theupper Alto Mayo district, whereas eastward the trait becameless frequent, until absent in the lower end of the Bajo Mayo.Bitterness was neither detected in any other collections ofthat survey nor has it been reported elsewhere to our knowl-edge in any other wild or cultivated forms of the species. Thepresence of the bitter trait stimulated a search for theresponsible fruit component(s).Lycopersicon spp. are known to harbor a-tomatine as the

characteristic glycoalkaloid ofthe genus (2). Although minuteconcentrations or none has been reported previously in ripetomato fruits, it was considered a potential source of thedetected bitterness. It is also known in Solanum spp; in fact,it is the only glycoalkaloid in Solanum pinnatisectum andSolanum polyadenium (3). Since a-tomatine is the prevailingform, and variants with fewer monosaccharides are rarely

detected and may be intermediates of biosynthesis or degra-dation or products of extraction (2), the glycoside is desig-nated hereinafter simply as "tomatine."Tomatine is of interest in respect to (i) toxicity to certain

fungi, thereby serving as a potential source of resistance toplant pathogens (4); (ii) repellance or toxicity to certainarthropods, thereby potentially serving as a defense com-pound (4, 5); (iii) source of steroids for synthesis of sterols(6); and (iv) toxicity to vertebrates.

MATERIALS AND METHODSStocks and Cultural Methods. The stocks utilized in this

research, which are listed in Table 1, were provided by theTomato Genetics Resource Center at the University of Cal-ifornia, Davis.The aforementioned accessions were grown in both field

and greenhouse in standard horticultural fashion. For thegenetic studies, hybridizations were made by conventionaltechniques. Automatic self-pollination was adequate for pro-duction of F2 and stock seed of parent lines under fieldconditions, but hand-pollination was necessary for fruit set inthe greenhouse.For measuring tomatine during fruit development, 6-10

plants each of the bitter LA2213, sweet LA2295, and cv.VF36 (LA490) were grown to maturity in May-June 1993under uniform greenhouse conditions. Flowers were self-pollinated and appropriately tagged at 10-day intervals untilfruits of the earliest series were fully mature. At that time allfruits of the experiment were simultaneously harvested,samples were extracted, and tomatine was assayed accordingto procedures described below. Sectors of 8-10 fruits eachwere massed for each genotype/age group.Sample Preparation. Leaf tissue was collected and dehy-

drated by two systems. Fresh leaves were either placeddirectly into a 60°C sample dryer or frozen and lyophilized.The dried samples were blended to a fine powder. Weighedfruit samples were sliced, placed in 60-ml polypropylenevials, microwaved, microblended to homogeneity, and ly-ophilized. Fruit and leaf powders were stored at -20°C untilanalyzed.The following simplified extraction procedure yielded valid

and consistent results. Place 50-mg samples of the prepareddehydrated leaf or fruit powder in 13 x 100 mm Teflon-capped culture tubes. Add 5.0 ml of 3.0 M HCI, 200 ,ul ofcholestane internal standard (100 ,g/ml of chloroform), and1-1.5 ml of chloroform to the culture tube and disperse in aVortex thoroughly. Hydrolyze tomatine to tomatidine andsugar moiety components in an 80°C waterbath for 3 hr. Aftercooling to room temperature, centrifuge to separate phases.Remove most of aqueous phase by aspiration. Add 2 ml ofwater and aspirate. Extract organic phase by adding 2 ml ofsaturated NaHCO3 solution, spinning in a Vortex, centrifug-

Abbreviation: BC, backcross.tTo whom reprint requests should be addressed.

12877

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ing to separate phases, and aspirating off most of the aqueousphase. Finally, wash the chloroform twice with water toremove residual salt. Transfer the organic phase to an au-tosampler vial. Since acylation and silanization did not affectquantitation of tomatidine, they were not applied to oursamples.GC. Analyze tomatidine levels by injecting 2-,ul sample

onto a 15-m DB-5 fused silica column (J & W Scientific,Folsom, CA) with 0.32-mm i.d. and 0.25-,um film thickness,fitted to a Varian 3700 gas chromatograph. The system wasequipped with a splitless 270°C injector and flame ionizationdetector (300°C). The system was operated using a 45-ssplitless period with H2 as the carrier gas (20 psi; 1 psi = 6.89kPa). The column temperature was programed from 200°C to275°C at 10°C min after a 1-min hold period. Routine periodicdeterminations were made of a reference solution consistingof 20 ,ug of cholestane and 60 ,ug of tomatidine per ml ofchloroform. For 145 such determinations, the overall CV was11.4%. Standardization tests of reference solutions in whichtomatidine concentrations were varied, the determinationswere linear with concentration (x = 0.33y; r = 0.998; P <0.01). No tomatidine was detected in nonhydrolyzed sam-ples. Cholestane, tomatidine, and tomatine were supplied bySigma.The following GC/MS procedure was utilized for verifiL-

cation of results obtained by the aforementioned protocol.GC/MS. Samples were analyzed by use of a Hewlett-

Packard 5890 gas chromatograph (Hewlett-Packard) inter-faced to a VG Trio-2 mass spectrometer (VG Masslab,Altrincham, U.K.). Helium was used as carrier gas at a linearvelocity of 35 cm/s. Splitless injections of 2 ,l were madeonto a 30-m DB-1 capillary column (J & W Scientific) of 0.25mm i.d. and 0.25-,m film thickness. The injector of the gaschromatograph was held at 285°C and the column tempera-ture was programed from 150°C (1 min hold to 300°C at10°C/min. Mass spectra were obtained using 70-eV electronionization over the range of m/z 40-650 every 1.0 s. Identi-fication of tomatidine in tomato extracts was confirmed fromchromatographic elution and mass spectra that matchedthose of a tomatidine standard. Other compounds presentsuch as tocopherols and sterols were identified by theircharacteristic mass spectra. Quantitative analysis was per-formed by comparing the integrated peak area from the totalion chromatogram from the area of a known amount ofcholestane, which was added as internal standard.

RESULTS

Preliminary analyses were made of field-grown material in1990. The bitter accessions LA2213 and LA2262 were com-pared with the sweet line LA2295 (Table 1). Ripe fruit of theformer yielded values of 913, 953, and 1267 ug of tomatidineper g of dry weight vs. 11 in the latter-an =100-folddifference. Leaf content-in the 2400-4900 range-did notdiffer appreciably between sweet and bitter line. Thus,whereas our determinations of the non-bitter lines corre-

spond with those reported by other workers for tomatocultivars, those of the Alto Mayo lines far exceed previouslyreported values for tomatine content in ripe fruits of L.esculentum.

Tests made on the same samples by GC/MS (Table 1)confirmed that the peaks measured in preliminary assayswere indeed those of tomatidine and also yielded comparablequantitative values except that no tomatidine was detected inthe sweet LA2310 line.Gas chromatograms of bitter and sweet lines are presented

in Fig. 1, exhibiting typical differences between them intomatine (measured as tomatidine) content. These differ-ences were maintained consistently throughout our later(1992 and 1993) assays under both field and greenhouseconditions, attended by variation in temperature, day length,and other factors. The GC and GC/MS analyses revealednumerous other peaks in addition to that of tomatidine. Overthe many bitter lines tested, the other peaks did not vary inproportion to levels of tomatidine, and, according to the MS,are of various tocopherols and sterols.The 1992 tests included assays of mature leaf content of

bitter and sweet parents and their F1 hybrid. All values werein a much higher range than those ofthe preliminary survey-17,000-31,000 ,ug/g of dry weight-corresponding with val-ues reported in the literature (7). Since the bitter line regis-tered lowest and the sweet line highest, leaf content does notreflect the enormous differences detected in ripe fruits-results that are concordant with our preliminary determina-tions.

In an attempt to assay variability of tomatine content in thebitter LA2213 line, we assayed tomatine of single fruits in thefollowing fashion. Three ripe fruits from each of six plants ofthis accession growing in the greenhouse were harvested inMay 1993, extracts were prepared, and GC values weredetermined for each fruit. The data treated as a 3 x 6 set byanalysis of variance yielded a mean of 4199, s = 1790, and a

Table 1. Accessions, sampling data, and tomatine determinations in L. esculentumTomatine,t ug/g of dry weight

GC

Pedigree no. Accession no. Source Tissue* Flavor Preliminary Standard GC/MS90L3473 LA2213 Nueva Cajamarca Leaf 4948 5080

Fruit Bitter 913 63001267 5080

90L3694 LA2262 La Huarpfa Leaf 2427Fruit Bitter 953 1690 1710

90L3736 LA2310 Pto. Santa Cruz Leaf 3684Fruit Sweet 11 10 0

92L6797 LA2213 Nueva Cajamarca Fruit Bitter 124151132713

92L6798 LA2295 Tarapoto Fruit Sweet 3992L6799 LA490 cv. VF36 Fruit Sweet 2992L6798 F1 LA2213 x LA2295 (bitter x sweet) Fruit Sweet 2592L6801 F1 LA2213 x LA490 (bitter x sweet) Fruit Sweet 38

All material was field-grown.*All sampled fruit were ripe.tDetermined as tomatidine.

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Proc. Natl. Acad. Sci. USA 91 (1994) 12879

A

0

0A0.

I_

4- BCholestane

Tomatidine

Jl jlB m~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

e Antioquia

COLOMBIA

* Valle

Retention time

FIG. 1. Gas chromatograms from extracts of ripe tomato fruits.(A) LA2295 normal (sweet) line. (B) LA2213 variant (bitter) line.

between-plant nonsignificant variance of 1100 and CV =42.6%. The lack of significant variance between plants en-dorses the uniformity of tomato inbred lines, but the excep-tionally high between-fruit variance argued for massing fruitsin our samplings. On the basis of such results, we slicedsectors of appropriate dimension from 6-10 fruits (often thepractical limit) for preparing samples. Even with this proce-dure, a relatively high error variance could be anticipated.Geographic Distribution. For the distribution of the high-

tomatine trait, we analyzed ripe fruit of 88 accessions fromthe whole distribution of Andean var. cerasiforme. Thisgroup sampled 56 accessions at random (=50% of the col-lections) from the Mayo district since it is ofprimary interest.The remaining accessions sample the rest of the distribution(Fig. 2). Low tomatine levels, typical of L. esculentum ingeneral, were found throughout all of the latter group; highreadings were restricted to the Mayo area. Within the latterdistrict, high tomatine prevailed in the Alto Mayo, diminish-ing to a low frequency at the eastern end of the Bajo Mayo(Fig. 3).As revealed by the survey of the Rfo Mayo drainage, the

distribution of high tomatine corresponds closely to that ofbitterness encountered in the previous survey (1). Accord-ingly, we analyzed the data by dividing samples into the fourpossible combinations of high vs. low tomatine; bitter vs.sweet. The latter determinations had been evaluated bytasting when all collections were planted together in a singletest plot in 1981-11 years before tomatine content wasassayed. In this breakdown, lines in which any degree ofbitterness could be detected were classified as bitter, and 500,ug/g (the low point in the distribution of tomatine concen-trations) divided low from high tomatine content. Thusclassified, the data for the survey were treated as a 2 x 2contingency table with the following distribution: bitter, 35vs. 1 (high vs. low tomatine); sweet, 8 vs. 44 (high vs. lowtomatine). For this array, the coefficient of association (Q) (8)is +0.99; the deviation from random assortment is highlysignificant (X2 = 54.37; 1 df, P < 0.001). Except for ninesamples (10%), association between the two variables wouldbe perfect.

Fruit Development. Results of the developmental study aredepicted in Fig. 4. They illustrate the general trends, theslight blips probably reflecting experimental error expectedfrom small fruit samples. In terms of either concentration of

BRAZIL

PERU

BOLIVIA

E. Puno

Arequipa £% 4~ch :bamba

FIG. 2. Distribution of sampled accessions in the Andean terri-tory ofL. esculentum var. cerasiforme. Bitterness and high tomatinewere found only in the Rio Mayo district.

tomatine or total content per fruit, the values for bitter andsweet lines coincide in starting at exceedingly high values(several times higher than those for foliage), descendingrapidly until the midperiod, when fruits have reached maturesize, at 30 days after pollination. Values for the large (=150g) fruited cv. VF36 remained consistently lower during thisperiod. In all lines, content per fruit increases during theinitial period and then remains fairly constant until fruitsreach mature size at =40 days, rapid enlargement beingresponsible for the sharp drop in concentration. Thereafteruntil fruits ofthe sweet lines are fully ripe, concentrations andcontent per fruit diminish slowly to near zero. From themidpoint until maturity at 50-60 days, the two sweet linesslowly decrease to essentially zero, the change necessarilyresulting from degradation. In these respects, our resultsagree with those of Eltayeb and Roddick (9), particularly inrespect to the values for cv. VF36, which, like their lines, islarge-fruited. Since our research and that of others detectedlittle or no tomatidine in ripe tomato fruit, degradation mustclearly proceed beyond the cleavage of tomatine into thecomponent sugar moiety and tomatidine.The point of special interest in the second period is the

pattern of tomatine levels in the bitter line, which remain

e. JufninE. Avacucho

E. Cusco

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12880 Agricultural Sciences: Rick et al.

7 ".LORETO

El Mirador o %*

Joqibilla oBaa0 p0 a'ran lo ,

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KEY ' oo acaisapaO Normal La Huarpia* Bitter Km 5700 High tomatine ,0

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essentially constant at the high level of 4000-5000 ,g/gthroughout. In this respect, the behavior is radically differentfrom that of the sweet lines, as described above. For theaforementioned reasons, this behavior cannot reflect changesin fruit size. Consequently it must be concluded that for thesecond period the difference between the lines is the conse-quence oftomatine catabolism in the sweet (normal) lines andthe lack thereof in the former.

Inheritance. During the 1992 field season, we tested theparental bitter (LA2213) and sweet (LA490, LA2295) lines, therespective bitter x sweet F1 hybrids, and segregating gener-ations. These populations were grown together in a compactfield area where conditions of soil, moisture, and other envi-ronmental factors were uniform. For parents and F1 hybrids,fruits were mass-harvested from each family; for segregatingpopulations, 10-20 fruits were massed from each plant. Asecond planting ofthe same lines (from the same seed lots) wasmade in Spring 1993 in an air-conditioned greenhouse.The assays on the 1992 material of parental lines were

comparable to those of 1990 (Table 1). Repeated analyses ofthe F1 families yielded low values in the range of the sweetparents, indicating complete recessivity of the high-tomatinetrait. For this reason, the most useful data would be yielded

70

60

iJ40z

0~-' 20

10

010 20 30 40 50 60

DAYS POST POLLINATION.3. LA2295 (Sweet) 6-LA490 (cv. 36) e- LA2213 (Bitr)

FIG. 4. Tomatine levels in fruits at different developmental stagesfrom normal (LA490, LA2295) and high-tomatine (LA2213) acces-sions.

FIG. 3. Distribution of normaland high tomatine, bitter andsweet accessions in the Rfo Mayodistrict of northern DepartmentSan Martfn, Peru.

by backcross (BC) to the bitter parent, no segregation beingexpected in BC to the sweet parents.

All of the segregating progenies that were analyzed werederived from hybridization between LA2213 and LA2295.Both ofthe segregating generations were grown in the field aswell as in the greenhouse.

Results for BC and F2 are presented in Fig. 5. The two BCgroups present similar images of segregation except thattomatidine readings tended to be higher in greenhouse plant-ings. The latter were higher by a factor of 1.63; for readingsof the bitter parent, the factor is 2.22. To combine the twogroups, the field readings were adjusted by a factor of 2.0.Thus represented in Fig. 5, the combined BC distribution for46 plants reveals two large groups for segregants with <100,g/g (28 plants) and >2000 (13 plants), the remaining 5 withintermediate levels. The distribution in the 43 greenhouse-grown F2 plants (Fig. 5) is similar in respect to grouping ofsegregates, with 31 in the lowest concentration, 5 in thehighest, and 7 intermediate. If genetic determination is qual-itative, as the F1 suggests, the division between high and lowclasses is to some extent arbitrary. A division at 500 wasselected on the basis of the distribution shapes and theaforementioned geographic data.On this basis, analysis of BC data yields totals of 28 low: 18

high-a deviation of 5 from monogenic expectation and 7.5from digenic (duplicate inheritance) hypothesis. The x2 cor-responding to the former is 2.17 (P = 0.2). For the F2, thesegregation is 34 high vs. 9 low, the deviation from mono-genic expectation being 1.75 with an associated x2 of 0.38 (P> 0.5); that from digenic, 6.3 with x2 = 39.82 (P < 0.001).Although recessives tend to be deficient, the fit is thereforecloser to monogenic expectation in both segregations. Thenature of genetic determination of the intermediates is un-clear but probably reflects action of a gene(s) of minor effecton recessive segregants.

DISCUSSIONInheritance. No references were found for high concentra-

tions of tomatine in ripe fruits comparable to those foundhere. Investigation of leaf tissue in hybrids between a lowparent, tomato cv. VF145B-7879, and a high parent, var.cerasiforme and derived segregating generations, led to theconclusion that codominant alleles segregating at one locuswere responsible (10). Similar studies of glycoalkloids ofvegetative tissues of cultivated and wild potatoes reveal that

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30

20

u

J-5U

30

20

10

BC

Z1 __ 1_. 1X t.: + -.

500 100O 1500 2000+

F,

500 1000 1500

Tomatine, ztg/g2000+

FIG. 5. Frequency distributions of segregation for concentrationsof tomatine in ripe fruits. Populations derived from cross of normal(LA2295) x high-tomatine (LA2213). (Upper) BC to high line.(Lower) F2-

the high levels of such compounds in the wild species aregenerally determined by two or more recessive genes (11). Ina similar investigation on the presence vs. absence of severalglycoalkaloids in strains of the wild potato relative, Solanumchacoense, presence was found to be inherited as dominantto absence, although total content of all glycoalkaloids of theF1 hybrid was closer to the low parent than to the high (12)and in that respect concordant with the preceding results (11).Any disagreement between these reports on leaf content andthe present results on ripe fruit is scarcely more surprisingthan the lack of relationship between them found in suchother traits as carotenoid content.

Toxicity. An aspect of consumer interest raised by thisstudy relates to the risks of consuming tomatoes with suchhigh tomatine content. Tomatine is commonly consideredless toxic to mammals than solanine, chacoene, and othersolanaceous alkaloids. We have not conducted studies oneffects ofconsuming ripe fruits of the investigated bitter lines;however, the following observations are relevant. First, inthe Alto Mayo, where all tomatillos have high tomatinecontent, they were consumed in considerable volume withoutapparent ill effect. A large usage was evident from ourobservations of harvests of plants growing mostly as spon-taneous weeds in the wild, fields, and gardens and from thevolume of fruits moving through the markets, notably themercado at Moyobamba, the departmental capital, and frominformation gained from conversations with many residents.Although the ripe fruits are generally prepared in soups andstews, cooking does not modify tomatine, as it is known to bestable at high temperatures. Bitterness did not seem to beobjectionable; in fact, we were not aware of it at the time ofcollecting; it might possibly be masked in the local recipes.Second, sampling ripe fruits in our tests had no adverseeffects. Repeated tasting was done of fresh fruit produced in

the experimental fields at Davis by one of us (C.M.R.) andverified by others; in the course of daily tasting a fairly largevolume of bitter accessions would be eaten without indiges-tion or other discomfort typical of solanaceous alkaloidpoisoning. These experiences are reminiscent of the con-sumption in certain regions of "fried green tomatoes" with-out manifestations of toxicity.Other Considerations. Finally, we face the question of the

significance of the bitter genotype. As explained, it is limitedto a tiny enclave in the total distribution-natural or inva-sive-of var. cerasiforme. Its genetic determination is totallyrecessive and relatively simple, probably monogenic. It isalso likely that the mutant DNA is defective, thus failing tocode for the normal enzymatic degradation of tomatine tobiologically inert compounds during maturation. An adaptivevalue is not evident, although subtle advantages might eludedetection. As in Lycopersicon, degradation ofglycoalkaloidsduring fruit maturation is a common phenomenon in theSolanaceae. It has been known in the tomato since at least1956 (13) and has been reported in various Solanum spp. (14,15). Toxic levels in foliage and unripe fruit can avert preda-tion by insects and other animals, but at fruit ripening, lossoftoxicity, along with increased sugar content, appearance ofcolor, odor, etc., would render the fruit edible and moreattractive to predators, thereby promoting seed dispersal. Inthis context, retention of high levels of alkaloid at maturationas in our bitter lines would not be adaptive. Thus, mutationto a biotype that lacks the capacity to degrade tomatine wasprobably a sporadic, random event of doubtful evolutionarysignificance. Human preference for bitter fruit or inhibitionby the alkaloid of bacterial decomposition of ripe fruit areconceivable but highly speculative adaptations. The mutationis probably a recent event that occurred in the area of itspresent distribution. We conclude on the basis of availableevidence that this variant originated from chance mutation ofthe normal allele coding for a tomatine-degrading enzyme.The bitter genotype might therefore facilitate investigationson the nature of tomatine catabolism.We thank Randolph Harney for vital technical assistance; Dr.

Lawrence Rappaport for use of his laboratory and equipment; andDr. Donald Nevins, Dr. Dina St. Clair, and Dr. Shang Fa Yang forlending essential equipment. The Tomato Genetics Resource Centerstaff assisted in growing progenies in the field and greenhouse.Critical reviews of the manuscript by Dr. E. Conn and Dr. J. Juvikare also highly appreciated.1. Rick, C. M. & Holle, M. (1990) Econ Bot. 44, 69-78.2. Schreiber, K. (1968) in The Alkaloids: Chemistry and Physiol-

ogy, ed. Manske, R. H. F. (Academic, New York), Vol. 10,1-192.

3. Gregory, P., Sinden, S. L., Osman, S. F., Tingey, W. M. &Chessin, D. A. (1981) J. Agric. Food Chem. 29, 1212-1215.

4. Roddick, J. G. (1986) in Solanaceae: Biology and Systematics,ed. D'Arcy, W. G. (Columbia, New York), pp. 201-222.

5. Juvik, J. A., Stevens, M. A. & Rick, C. M. (1982) Hort. Sci.17, 764-766.

6. Fontaine, T. D., Schaffer, P. S., Doukas, H. M., Scott, W. E.,Ma, R. M., Turkot, V. A., DeEds, F., Wilson, R. H. & Doo-little, S. P. (1955) U.S. Agric. Res. Ser., 73-78.

7. Roddick, J. G. (1974) Phytochemistry 13, 9-25.8. Yule, G. U. & Kendall, M. G. (1950) An Introduction to the

Theory of Statistics (Griffen, London).9. Eltayeb, E. A. & Roddick, J. G. (1984) J. Exp. Bot. 35,

252-260.10. Juvik, J. A. & Stevens, M. A. (1982) J. Am. Soc. Hort. Sci.

107, 1061-1065.11. Ross, H. (1966) Am. Potato J. 43, 63-80.12. McCollum, G. D. & Sinden, S. L. (1979) Am. Potato J. 56,

95-113.13. Sander, H. (1956) Planta 47, 374-400.14. Pandeya, S. C., Sarat Babu, G. V. & Bhatt, A. B. (1981)Planta

Medica 42, 409-411.15. Telek, L., Delphin, H. & Cabanillas, E. (1977) Econ. Bot. 31,

120-128.

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