Chronic Cassava Toxicity

Home , Lotaustralin

4/63 7L ARCHIV NESTEL C-010e 26528 II Chronic Cassava Toxicity

Proceedings of an interdisciplinary workshop London, England, 29-30 January 1973

Editors: Barry Nestel and Reginald Maclntyre

INTERNATIONAL CENTRE DE RECHERCHES DEVELOPMENT POUR LE DEVELOPPEMENT RESEARCH CENTRE INTERNATIONAL

Oawa, Canada 1973 IDRC-OlOe

CHRONIC CASSAVA TOXICITY

Proceedings of an interdisciplinary workshop London, England, 29-30 January 1973

Editors: BARRY NESTEL AND REGINALD MACINTYRE

Foreword Barry Nestel 5-7 Workshop Participants 8-10 Current utilization and future potential for cassava Barry Nestel 11-26 Cassava as food: toxicity and technology D. G. Coursey 27-36 Cyanide toxicity in relation to the cassava research program of CIAT in Colombia James H. Cock 37-40 Cyanide toxicity and cassava research at the International Institute of Tropical Agriculture, Ibadan, Nigeria Sidki Sadik and Sang Ki Hahn 41-42 The cyanogenic character of cassava (Manihor esculenta) G. H. de Bruijn 43-48 The genetics of cyanogenesis Monica A. Hughes 49-54 Cyanogenic glycosides: their occurrence, biosynthesis, and function Eric E. Conn 55-63 Physiological and genetic aspects of cyanogenesis in cassava and other plants G. W. Butler, P. F. Reay, and B. A. Tapper 65-71 Biosynthesis of cyanogenic glucosides in cassava (Manihot spp.) Frederick Nartey 73-87 Assay methods for hydrocyanic acid in plant tissues and their application in studies of cyanogenic glycosides in Manihot esculenta A. Zitnak 89-96 The mode of cyanide detoxication 0. L. Oke 97-104 Chronic cyanide toxicity in domestic animals D. C. Hill 105-111 Implications of cyanide toxicity in animal feeding studies using high cassava rations Jerome H. Maner and Guillermo Gômez 113-120 Cyanide and human disease J. Wilson 121-125 Ataxic neuropathy associated with high cassava diets in West Africa B. 0. Osuntokun 127-1 38 Endemic goitre and high cassava diets in eastern Nigeria 0. L. Ekpechi 139-145 Evidence of an antithyroid action of cassava in man and in animals F. Delange, M. van der Velden, and A. M. Ermans 147-151 Mechanism of the goitrogenic action of cassava A. M. Ermans, M. van der Velden, J. Kinthaert, and F. Delange 153-1 57 Summary of the General Discussion 159-162 Physiological and Genetic Aspects of Cyanogenesis in Cassava and Other Plants

G. W. BUTLER, P. F. REAY, AND B. A. TAPPER Department of ScienlJlc and Industrial Research Applied Biochemistry Division Palmerston North, New Zealand

BUTLER, G. W., P. F. REAY, AND B. A. TAPPER. 1973. Physiological and genetic aspects of cyanogenesis in cassava and other plants, p. 65-71. In Chronic cassava toxicity: proceedings of an interdisciplinary workshop, London, England, 29-30 January 1973. mt. Develop. Res. Centre Monogr. IDRC-OlOe.

AbstractAvailable data on the pathways for degradation of cyanoglucosides and subsequent fate of the breakdown products in cassava (Manihot spp.) and other plants are discussed. Also considered is the degradation of cyanoglucosides after ingestion by animals and parasitic organisms. The physiological and genetic factors which give rise to variations in cyanoglucoside content in plants are also discussed.

Résumé Nous examinons de facon critique nos connaissances sur les voies metaboliques de degradation des cyanoglucosides et le sort subsequent des produits qui en résultent chez le manioc (Manihot spp.) et autres plantes. Nous considérons egalement la degradation des cyanoglucosides aprés ingestion par les animaux et par les organismes parasites. Enfin, nous examinons les facteurs physiologiques et genetiques responsables des variations de Ia teneur en cyanoglucosides des plantes.

Degradation of Cyanoglucosides activity catalyses the dissociation of the aglycone to hydrogen cyanide (HCN) and benzaldehyde WHEN plant tissues containing cyanoglucosides (hydroxynitrile lyase synonymous with oxyni- are crushed or autolysed, an enzymic hydrolysis trilase below). Emulsin is specific for-glucosides takes place releasing the sugar moiety and the and both a- and fl-galactosides. As well as being aglycone. The crushing of the plants probably specific for the sugar moiety, it shows specificity allows the glucosidase and glucoside to diffuse foraromatic cyanogenic glucosides,sinceit together and react. The enzymes hydrolysing these hydrolyses linamarin and lotaustralin very slowly glycos ides are fl-glucosidases with differing degrees (Butler et at. 1965). Emulsin will also hydrolyse of specificity for the aglycone portion of the com- noncyanogenic glucosides such as arbutin and pound. salicin. The enzyme system emulsin, isolated from The glucosidase, linamarase, isolated from linen almond kernels, has been reported by Haisman flax seed, hydrolyses both aromatic and aliphatic and Knight (1967) to have at least three separate cyanogenic glucosides, but not diglucosides such enzymic activities against cyanogenic glucosides. as amygdalin (Butler et at. 1965). Arbutin and The first converts the diglucoside amygdalin to salicin are also hydrolysed at appreciable rates. the monoglucoside prunasin (amygdalin lyase), The linamarase extracted from clover leaves could which is hydrolysed by the second to give the not be purified by the techniques described for aglycone and glucose (prunasm lyase). The third linseed linamarase, because of problems of enzyme

65 66 MONOGRAPH IDRC-OIOe stability, and its substrate specificity is not known. action, whereas grating will result in maximal Nartey (1968) showed that a crude preparation tissue damage and hence maximal HCN liberation. from cassava leaves showed strong activity against An important additional aspect to consider is linamarin and lotaustralin, mild activity againstthe extent to which HCN will be retained as salicin, and weak activity against fl-methyl glyco- cyanhydrins by reacting with carbonyl groups in side and amygdalin. Hughes (198a) studiedvarious compounds, especially carbohydrates. fl-glucosidase production in callus tissue from Cassava root tissues contain appreciable amounts white clover stems. Evidence was obtained from of hexoses (about 4% of the dry matter; Ketiku Michaelis constants that two distinct fl-gluco-and Oyenuga 1970) and it can be assumed that sidases were produced: a "low-activity" fl-gluco- cyanhydrins would be readily formed. Linamarin sidase with a low Michaelis constant and a "highand lotaustralin are not especially acid-labile, and activity" type with a high Michaelis constant. the statements in the literature on the lability of The latter activity was due to linamarase, since linamarin (Dunstan and Henry 1903, 1906; Collard no "high-activity" extracts were obtained from and Levi 1959; Wood 1966) can be explained in callus tissue from linamarase-negative genotypes. terms of the lability of such cyanhydrins. Where In studies on DEAE-cellulose fractions from other than fresh plant tissueis analysed, the clover leaves, further evidence for differences in probability that cyanhydrins are present as a fl-glucosidase specific activity between linamarase-result of cyanoglucoside degradation should be positive and linamarase-negative genotypes was kept in mind. In this connection, the recommenda- obtained (Hughes l968b). tion to add glucose to cassava products to avoid Although the aglycones formed after hydrolysis cassava toxicity can only be partially effective, of the glycoside reversibly dissociate to HCN and since the glucose cyanhydrin would be dissociated aldehyde or ketone, the reaction is catalysed inin the intestine with absorption of the cyanide plant tissues by oxynitrilases which are most into the bloodstream. de Bruijn (1971) discussed active atpH values where the nonenzymic reaction this and showed that glucose additions to cassava is slow (Conn 1969). The reaction proceeds to root macerates scarcely reduce HCN output. It completion at physiological pH values, is more seems important to us to establish the extent to rapid at alkaline pH values, and is also catalysed which cyanhydrins are formed during preparation by cations and amines. The pH optimum of the of cassava food products and to study ways where- oxynitrilases from sorghum and from bitter almond by their production is minimised. kernels lies between Sand 6(Bové and Conn 1961), Where cyanoglucosides are ingested by rumi- apH at which the nonenzymic dissociation is slow. nants, fl-glucosidases from the rumen microflora The oxynitrilase activity would thus be more will also readily hydrolyse the glycosidic bond important in tissue extracts whose pH was below liberating HCN (Coop and Blakley 1949). The neutrality. hydrogen cyanide released in the rumen is rapidly The enzymic activitiesbringing about the absorbed through the wall of the rumen into the hydrolysis of cyanogenic glucosides are usually blood stream, flowing to the liver where cyanide is present in such quantity and are so active that a detoxified with the formation of thiocyanate. The very rapid breakdown of cyanogenic glucosiderate of detoxication by liver tissue in vitro was results in crushing or damaging the tissue. The fast enough to account for most of the cyanide rapid breakdown of cyanoglucosides by endo- in the rumen of the animal. genous enzymes is an important factor in the The minimum lethal dose of hydrogen cyanide toxicity of these plantsto mammals and to for sheep is 2.4 mg/kg (Coop and Blakley 1949), pathogenic or parasitic organisms, and these in agreement with values found for other animals. aspects will now be considered in turn in relation The minimum lethal dose of lotaustralin: linamarin to cassava. is 4.5 mg HCN/kg body weight; the tolerance to a With respect to cassava toxicity to humans, higher dose can be ascribed to the time required there will clearly be marked differences in the for consumption of the feed and to the time re- extent oflinamarase action on the cyanoglucosides, quired for release of cyanide by linamarase, which depending on the amount of tissue damage during may be slower in the rumen because of dilution. preparation of the cassava. Peeling off the rind Over long periods, sheep were able to detoxify will cause minimal tissue damage and linamarase and tolerate 3.9 mg HCN/kg per hour for many BUTLER ET AL. CYANOGENESIS IN CASSAVA 67 hours. No evidence of adaptation to HCN was mechanism is absolute and so we can consider noticed. cyanogenesis only in comparative and not in Where the ingestion of forage is relatively slow, absolute terms as a defensive character. It may not as in the normal grazing situation, sheep could be efficient, but it may well be enough to deter well tolerate 15-20mg HCN/kg body weight per many would-be grazers and parasites." day (Coop and Blakley,1949).Since clover With respect to the metabolism of HCN in plant makes up about 60% of a pasture and the level of tissues, it has been demonstrated in most plants glucoside in the leaves is three times that in the studied that HCN can be incorporated into aspara- petioles which make up 40% of the plant, a critical gine, with the nitrile group becoming the amide level of cyanide in the leaves would be 3500 ppm group of asparagine (see review by Conn and (dry weight) if the sheep ate 1.5 kg dry weight/day. Butler 1969). In two cyanogenic plants, it has been Corkill (1952) bred a strain of white clover with deduced from radioisotope studies that '4C label HCN levels approaching 3500 ppm, but no evi- from biosynthesised cyanoglucoside can be trans- dence of toxicity to grazing sheep was observed, ferred to asparagine, presumably by degradation nor were cyanogenic strains less palatable than of the cyanoglucosides and reassimilation of the acyanogenic clover strains. liberated HCN into asparagine (Abrol and Conn In contrast to white clover, sorghum has often 1966 for Lotus spp.; Nartey 1969 for cassava). It caused death of grazing cattle and is regarded as thus appears that the cyanoglucosides are meta- dangerous to feed when the level in the leaves bolically active and that they "represent some exceeds 500 ppm (dry weight) (Boyd et al. 1938). form of storage carbon and nitrogen which are However, Rose (1941) recorded that cattle con- capable of being utilised by the plant" (Nartey tinuously grazing sudan grass containing up to 1969). The hypothesis thatthisrepresents a 1330 ppm HCN were unaffected, whereas dairy mechanism for recovering and recycling nitrogen cattle which ate rapidly when put onto sudan is an attractive one which needs further evalua- grass were affected by lower levels of cyanide. He tionthe alternative is to regard it as a detoxica- also noticed that sheep safely grazed sorghum tion mechanism rather than a central metabolic hybrids which had been poisonous to cattle. pathway, especially as the capability to form Since the maximum level of cyanide in clover asparagine from HCN also exists in plants which pastures might be as high as 1000 ppm, well above do not contain cyanoglucosides. a dangerous level in sorghum, it appears that the An additional mechanism for HCN detoxifica- latter might be more toxic than clover. This could tion in plants was reported recently by Chew and be explained by a faster hydrolysis of the glucoside Boey (1972), namely the presence of rhodanese in sorghum. Coop and Blakley (1949) noted that activityincrude extracts of cassava leaves. the presence of sugars greatly reduced the rate of Rhodanese catalyses the formation of thiocyanate hydrolysis of lotaustralin, so the quantity of free from free cyanide and a sulfur donor in animal sugar in the forage may also affect the toxicity by tissues and some bacterial species, but it has not retarding hydrolysis of the cyanoglucoside. previously been demonstrated in plant tissues. de Bruijn (1971) gives levels of HCN in leaves Chew and Boey calculated that sufficient rhodanese of 15 cassava clones ranging from 540 to 1090 activity was present in tapioca leaves to assimilate ppm (fresh weight), corresponding approximately any free cyanide released by cyanoglucoside to 2000-4000 ppm (dry weight), i.e. considerably hydrolysis in the cell. The significance of rhodanese higher than for sorghum and sudan grass and in the detoxication of cyanide in plant tissues in tending to be higher than observed for white relation to the alternative pathway to asparagine clover or lotus. described earlier requires further evaluation. The extent to which liberation of HCN represents a defence mechanism against insects and parasitic organisms should also be considered. Genetical Variation in Jones (1972) discussed this question and concluded Cyanoglucoside Content that "though several animals and plants are able to eat or parasitize cyanogenic plants, this does We have been considerably influenced by the not detract from the possible basic function ofrecent view of Jones (1972) in preparing this cyanogenesis as a defence mechanism. No defence section.Inseveralcyanogenic species,poly- 68 MONOGRAPH !DRC-OIOe morphism is exhibited with respect to cyanogluco- 20-fold) in cyanide content reported between side content and such species are of great interest clonal lines by many investigators. It would be to geneticists in relation to interactions between necessary for the acyanogenic line selected to be environment and genotype. Cyanogenic plants of the phenotypes lacking cyanoglucoside; it could contain both cyanoglucosides and the appropriate also be advantageous for the line to be positive for fl-glucosidase, but plants which are acyanogenic linamarase, since a high level of this enzyme would may differ in their cyanoglucoside and fl-gluco- be desirable if the line became contaminated in sidase composition, as follows: practice. A screening program of genotypes for Gross low or zero cyanoglucoside followed by clonal phenotype propagation of the selected material should be possible. Cyanoglucoside + enzyme Cyanogenic Cyanoglucoside + no enzyme Acyanogenic In considering whether breeding for acyano- Enzyme but no cyanoglucosidesAcyanogenic genesis is desirable, it seems important to dis- Neither cyanoglucoside tinguish between the requirements for subsistence nor enzyme Acyanogenic cultivation so widespread in the tropics and the prospects for more mechanised cultivation which Jones (1972) states that this scheme has been is developing with the increasing use of cassava shown for Prunus amygdalus. Sorghum vulgare, as an animal foodstuff. Whereas it may be neces- Trifolium repens, and probably for Sambucus sary to retain cyanoglucoside in the outer integu- nigra. He stated: "In 7'. repens (Williams, 1939;ment of the root for pest protection in the case of Corkill, 1940, 1949; Atwood and Sullivan, 1943) subsistence farming, the use of cassava in more and L. corniculatus (Dawson, 1941; Bansal, 1966) intensive agricultural operations might be accom- the presence of both the cyanogenic glucosides panied by alternative methods of protection against linamarin and lotaustralin is determined by aplant pathogens and parasites (systemic fungi- single dominant allele, while the presence of the cides, etc.). appropriate fl-glucosidase is also determined by It might be possible to breed a compromise with a single dominant allele, at a locus not genetically a level of cyanoglucoside in the outer integument linked to the glucoside one. With L. corniculatus which is sufficiently high to act as a defence against there is the added complication that the plantpathogens and parasites, but with a negligible behaves as an autotetraploid for theseloci cyanoglucoside content in the main part of the (Dawson, 1941; Bansal, 1966)." root. Cyanoglucosides are oftenfairlyrigidly The environmental factors influencing the fre- restricted to particular organs, e.g. roots of T. quency of cyanogenesis in T. repens have been repens have very low levels and in the passion- identified as temperature by Daday (l954a, b) fruit Pass ffiorum mollissima the cyanoglucoside is and diflrential eating by Jones (1962, 1966, 1970). present mainly in the rind and rarely in the seeds Daday has concluded that low temperaturesor the flesh. In our opinion, however, it would favour acyanogenic lines while Jones has placed seem preferable to breed for complete acyano- emphasis on the differential eating of cyanogenic genicity, mainly because of the wide variations in and acyanogenic plants by slugs and snails. cyanoglucoside content which might arise from In the case oT cassava, the balance betweenphysiological causes, even in plants bred for low cyanogenic and acyanogenic plants in the agri- cyanoglucoside content. cultural situations in which it is grown is tilted so An analogy could be drawn with the vigorous strongly toward cyanogenic plants that acyano- and successful plant-breeding program mounted genicity is not recognised. Perhaps the systems ofin Canada in recent years to markedly lower the clonal propagation employed agriculturally areglucosinolate content of rapeseed, and also to stabilising or perpetuating this situation. It does completely remove erucic acid which is a major seem likely, however, that a comprehensive screen- fatty acid constituent in the cultivated varieties of ing of cassava seedlings would reveal genotypes Brassica napus and B. campestris previously in use. which were acyanogenic. The report that anMany thousands of seedlings and seeds were acyanogenic line existed in Indonesian collections screened in this program and the program of but was lost during World War II lends support topurposeful plant breeding backed up by first-class this expectation, as do the wide differences (up to biochemistry and chemistry represents a first- BUTLER ET AL.: CYANOGENESIS IN CASSAVA 69 class example of how such a program should bebecause plants can adapt to short droughts by approached and carried through. Dr R. K. abscission of some leaves. Downey, plant breeder for the Canada Depart- ment of Agriculture in Saskatoon, heads the plant Mineral Nutrition breeding team responsible (see also Kondra and Stefansson 1970). In general it would be expected that mineral nutritional regimes which increase the pool of nonprotein-nitrogen within the plants, and in particular the valine and isoleucine pools, could Physiological Variation in lead to increased cyanoglucoside levels, provided Cyanoglucoside Content there were no direct inhibitory effects of the particular mineral balance or imbalance on the The literature on cassava reflects some per- biosynthesis of the cyanoglucosides. Thus a high plexity amongst investigators who have attempted nitrogen fertilising regime would tend to elevate to rationalise the variations in cyanoglucoside cyanoglucosides, as observed by de Bruijn (1971) content as influenced by physiological factors. and others. Similarly macro-element and micro- This is not surprising since the cyanoglucoside element deficiencies, which often result in the non- levels will be determined by a number of physio- protein-nitrogen pool being large, could well cause logical factors interacting with each other. elevated cyanoglucoside levels. On the lateritic soils on which cassava is so often grown, the fre- Variation with Age quency of such mineral imbalances seems likely de Bruijn's (1971) study showed that cyano- to be high, so that elevated cyanoglucoside contents glucoside levels are highest in young cassava leaves from this cause would be expected to be common. and petioles, and decline with age. This is a fairly Potash deficiency would seem to be a particularly widespread pattern in cyanogenic species. He likely cause from the literature we have seen. found no indications that the glucoside con- centrations of the tuberous roots are directly Effect of Shade and Ring-Barking related to plant age and considered that fluctua- Shading to 35 and 70% daylight for 8 weeks tions in glucoside content during growth are increased the cyanoglucoside content of leaves and mainly due to changes in ecological conditions. correspondingly decreased cyanoglucosidesin roots (de Bruijn, 1971), perhaps by reduction of Water Stress translocation to the roots either of cyanoglucosides or cyanoglucoside precursors. de Bruijn Darjanto obtained comparative figures(see (1971) showed that ringing of stems also in- Bolhuis 1954) for two cassava clones which were creased cyanoglucoside content in the bark above each grown on old laterite soil, on two nearby the incision. It would seem important to clarify sites. One site had regularly been used as a paddy the extent to which linamarin and lotaustralin are field; the other drier location had been used for synthesised in cassava root tissue and the extent, cultivation of upland rice and citronella grass, if at all, to which they are translocated from the and had lower fertility especially with respect to leaves. It may be that the roots, besides acting as signs of potash deficiency. The cyanoglucoside a "physiological sink" for carbohydrate, also content in roots from both clones were markedly behave similarly for cyanoglucosides which have increased (approximately three-fold) when grown been synthesised in the aerial portions of the plant. in the drier situation, but the apparent differences in soil fertility complicate interpretation. de Bruijn (1971) grew young plants in bags for 2 months, Conclusions with water regimes which were two-thirds and one-third optimal and observed an increase in A consideration of the complexity of the physio- cyanide content per unit dry matter in both roots logical interactions governing the cyanoglucoside and leaves with increasing dryness. However, he content of cassava leads us to the conclusion that states that in the field the glucoside content would agricultural practices to reduce the cyanide levels be increased only after a very long dry period, by physiological manipulations are not practicable, 70 MONOGRAPH IDRC-OIOe and that genetic manipulation is the only practi- BOYD, F. T., 0. S. AAMODT, G. BOI-ISTEDT, AND E. cable approach. TRUOG. 1938. Sudangrass managementfor control of cyanide poisoning. J. Amer. Soc. Agron. 30: 569-582. Appendix BRUIJN, G. H. DE. 1971.Étude du caractère cyo- génétique du manioc (Man ihot esculenta Crantz). The Goitrogenic Effect of Cyanogenic White Clover Wageningen. In work carried out at this station in the mid BUTLER, G. W., R. W. BAILEY, AND L. D. KENNEDY. 1965.Studies on the glucosidase 'Linamarase." 1950s, rats, guinea pigs, and sheep were fed Phytochemistry 4: 369-381. cyanogenic and noncyanogenic white clover (Flux BUTLER, G. W., D. S. FLUX, G. B. PETERSEN, E. W. et al. 1956: Butler et at. 1957). Goitrogenic effects WRIGHT, A. C. GLENDAY, AND J. M. JOHNSON. were observed in rats and guinea pigs and with 1957. Goitrogenic effect of white clover (Trifolium rats it was shown by periodic injections of thio- repens L.) II. New Zealand J. Sci. Technol. 38A: cyanate that the levels of thiocyanate encountered 793-802. in the clover-feeding experiments exerted a direct CHEW, M. Y., AND C. G. BOEY. depressing effect on thyroid gland activity as 1972.Rhodanese of tapioca leaf.Phytochemistry 11: 167-169. evidenced by incorporation of 13h1. Serum thio- COLLARD,P.,ANDS.Levi.1959 A two-stage cyanate levels in the two groups of rats were 6.4 fermentation of cassava. Nature 183: 620-62 I. mg/I 00 ml and 15.6 mg/I00 ml for noncyanogenic CONN, E. E.1969.Cyanogenic glycosides. and cyanogenic clover feeding respectively. J. Agric. Food Chem. 17: 519-526. In sheep-feeding experiments lasting 43 days, CONN, E. E., AND G. W. BUTLER. it was shown that serum thiocyanate levels rose to 1969.The biosynthesis of cyanogenic glucosides and other simple levels similar to those for guinea pigs and rats, but nitrogen compounds, p. 47-74. In J. B. Harborne relatively slight goitrogenic effects were observed, and T. Swain [ed.J Perspectives in Phytochemistry. namely a reduction in the total iodine per unit wet Academic Press, London-New York. weight of thyroid. In subsequent long-term grazing Coop, 1. E., AND R. L. BLAKLEY. 1949.The metabo- experiments where lambs were born from ewes lism and toxicity of cyanides and cyanogenetic grazing on pure cyanogenic clover, much larger glucosldes in sheep. I. Activity in the rumen. New goitrogenic effects were observed in the lambs Zealand J. Sci. Technol. 30A: 277-291. (Flux et at.1963) in that thyroid glands were CORKILL, L. 1940.Cyanogenesis in white clover significantly heavier than those of ewes grazed on (Trifolium repens L.) I. Cyanogenesis in single plants. ryegrass pastures. The goitrogenic effects were not New Zealand J. Sci. Technol. 22B: 65-67. sufficient to influence the productive performance 1949.Cyanogenesis in white clover (Trifolium of grazing animals, however. repens L.) V. The inheritance of cyanogenesis. New Serum thiocyanate levels in dairy cows (both Zealand J. Sd. Technol. 23B: 178-193. lactating and nonlactating) were low and a goitro- 1952. Cyanogenesis in white clover (TrfoIium genie effect from cyanogenic white clover on these repens L.) VI. Experiments with high-glucoside and animals appeared unlikely. glucoside-free strains. New Zealand J. Sci. Technol. 34A: 1-16. DADAY, H. l954a.Gene frequencies in wild popu- References lations of Trifolium repens. I. Distribution by lati- tude. Heredity 8: 61-78. ABROL, Y. P., AND E. E. CONN. 1966.Studies on 1954b.Gene frequencies in wild populations of cyanide metabolism in Lotus arabicus L. and Lotus Trfoliumrepens.II.Distributionbyaltitude. tenuis L.Phytochemistry 5: 237-242. Heredity 8: 377. ATWOOD, S. S., AND J. T. SULLIVAN. 1943.Inheri- 1965.Gene frequencies in wild populations of tance of a cyanogenetic glucoside and its hydrolysing Tr(folium repens. III. Mechanism of natural selection. enzyme in Trijolium repens. J. Hered. 34: 311-320. Heredity 20: 355-365. BANSAL, R. D.1966.Studies on procedures for DAWSON, C. D. R.1941.Tetrasomic inheritance in combining clones of birdsfoot trefoil, Lotus corni- Lotus corniculatus L.J. Genet. 42: 49-72. culatus, L.Ph.D. thesis, Cornell University, U.S.A. DUNSTAN, W. R., AND T. A. HENRY. 1903.Cyano- B0LHuIs, C. G.1954.The toxicity of cassava roots. genesis in plants. III. On phaseolunatin, the cyano- Neth. J. Agric. Sci. 2: 176-183. genetic glucoside of Pha.seolus lunatus. Proc. Roy. BovE, C., and E. E. 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