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A rapid and sensitive method to detect cyanogenesis using microtiterplates
Biochemical Systematics and Ecology, Vol. 19, No. 6, pp. 519-522, 1991. Printed in Great Britain.
0305-1978/91 $3.00+0.00 © 1991 Pergamon Press plc.
A Rapid and Sensitive Method to Detect Cyanogenesis Using Microtiterplates P. KAKES Faculty of Biology, Free University of Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Key Word Index--Cyanogenesis; cyanogenic glucosides; ~-glucosidase. Abstract--A method to test plant material for cyanogenesis (the production of HCN) is described. The test requires small amounts of plant material and uses only inexpensive and readily available materials. The method is particularly well suited for field studies. The method has been tested on 11 plant species, in a range of plant tissues.
Introduction Cyanogenesis, or the production of HCN, is a common phenomenon in the plant kingdom. Among the higher plants some 1000 species have been reported as cyanogenic. Very often the evidence rests on the study of a few individuals of one species. Detailed studies on a limited number of species have shown both continuous variation for the amount of HCN produced and polymorphism in the strict sense: the o c c u r r e n c e of cyanogenic and acyanogenic plants within one population. For a better understanding of the o c c u r r e n c e and significance of cyanogenesis it is important to study representative samples of populations, and to study different tissues at different times in the growing season. Although quantitative tests are indispensable, often a semi-quantitative test does give sufficient information. Such tests are generally better suited to study larger numbers of samples. We will describe a method that is quick (100 samples take less than 1 h of preparation time and can be processed within one day), requires small amounts of plant material and uses only readily available materials. Several authors have in the last few years published methods to detect cyanogenesis in plant material [i.e. Brimer and M~lgaard (1986), Brimer (1988), Nahrstedt e t a / . (1981), Almazan (1987)]. Reviews and comparisons of methods have been published by Brimer (1988) and Zitnak (1973); Hegnauer (1986) has discussed the advantages and disadvantages of the FeigI-Anger method (Feigl and Anger, 1966), compared to several other procedures to detect HCN. The method to be described in this paper is different from earlier ones in the following respects. (i) By using a small volume: 0.3 ml for standard microtiterplates--the diffusion time is shortened and the method rendered more sensitive, allowing the use of very small amounts of material. (ii) The use of FeigI-Anger test paper instead of the commonly used alkaline picrate paper has the advantage of a short reaction time. The blue spot is developed almost instantaneously, whereas the alkaline picrate reaction takes 24 h to complete at 28°C (Williams and Edwards, 1980). Short diffusion and reaction times ensure that the rate of colour development is dependent on the rate of HCN formation in the plant material, which is important in enzyme studies. Long incubation times (24 h and longer) are undesirable in view of the spurious positive reaction caused by bacterial contamination of the samples (Saupe eta/., 1982). (iii) The FeigI-Anger test is more specific than the alkaline picrate reaction, reducing the o c c u r r e n c e of false positives. (iv) In field studies the material can be collected directly in the wells of microtiter plates. In that way 96 samples can be transported and processed in a very small (Received 25 November 1990) 519
520
F~ KAKES
volume: 12x8x1.5 cm. (v) The method avoids the use of toluene and picric acid, both potentially hazardous chemicals.
Cyanogenesis and cyanotypes Plants that spontaneously produce HCN when the cells are damaged are called cyanogenic. Cyanogenesis is the result of the hydrolysis of cyanoglucosides by specific J3-glucosidases. It is presumed that cyanoglucosides are present in the vacuole; the corresponding 13-glucosidases have been shown to be located in the cell walls in two species: Phaseolus lunatus and Trifolium repens (Frehner and Conn, 1987; Kakes, 1985). In species monomorphic for cyanogenesis the plant usually contains both cyanoglucoside(s) and specific 13-glucosidase(s). In polymorphic species the acyanogenic genotypes or organs may lack the cyanoglucosides, the corresponding ~-glucosidase or both. In such cases, four cyanotypes may be distinguished by adding cyanoglucosides or a j]-glucosidase to the sample. If the nature of the cyanoglucoside(s) in the species is not known, it may be useful to add a broad spectrum j]-glucosidase to (part of) the sample. Helicase, a mixture of enzymes from the gut of Helixpomatia, may be used in this case. Cyanolipids are not hydrolysed by helicase, and some cyanoglucosides very slowly (Brimer, 1988) so samples that give negative results need further examination. Description of the Method The samples are placed on the bottom of the wells of a microtiterplate. Wells of 300 ~1 are suitable for leaves of Tr/fo//um repens and Lotus cornicu/atus, larger wells may be used for more voluminous or weakly cyanogenic samples. The microtiterplates are placed in a refrigerator at -20°C, for at least 1 h, After thawing, 100 p.I of distilled water is pipet-ted in each well, the surface of the plate is carefully wiped dry and a sheet of FeigI-Anger test paper (see later) is placed over the wells. The sheet is covered with a glass plate of the size of the microtiterplate and the complete sandwich is secured with paperclamps. The sandwich is placed in an incubator at 35°C. After 2-3 h, depending on the material, the paper is removed. Over wells with cyanogenic material blue spots will have formed. The minimum amount of HCN detected is approximately 40 nmol HCN per well. Up to 180 nmol HCN there is a clear relation between the amount of HCN and the intensity of the blue spot. Using leaf disks of a cyanogenic plant of Trifo/iurn repens, I found that 2 mg fresh weight was enough to obtain a faint blue spot. In Trifo//umrepens all four cyanotypes occur. They are easily recognized by complementing the leaf material with linamarin, one of the cyanogenic glucosides in Trifo//urn repens, and with linamarase, the corresponding ~-glucosidase. In this study plants of known genotype were used to test the method. Plants with linamarin, but lacking linamarase gave a clear positive reaction with 10 mU of linmarase, plants lacking linamarin but containing linamarase gave a positive reaction with 40 nmol of linamarin. Linamarin and linamarase may be purchased but relatively crude preparations of both substances give equally good results. Simple methods to prepare linamarin from flax seedlings and linamarase from linseed are described by Kakes (1987). By using known amounts of plant material (i.e. leaf disks) and comparing the spots with a graded series of HCN, the method, essentially qualitative, can be made semi-quantitative. The spots retain colour and intensity for several weeks if kept cool and in the dark. Testing of the method with different cyanogen/c substrates and testing for fa/se positive reactions. Table 1 shows cyanogenic species known to be cyanogenic from the literature, tested with our method. Different plant organs, both cyanogenic and acyanogenic are included. It is clear that the method can be used on a wide variety of species and has good discriminating power. The negative reaction of the leaves of Prunus/us/tan/ca is unexpected, as Rosenthaler (1919) found all parts of this plant cyanogenic. The polymorphism found in Trig/ochin rnar/tirna has to my knowledge not been reported earlier. As small amounts of material may be used, plant parts as leaflets, cut disks of leaves and small seeds may be used, thereby revealing individual and within-plant variation. Brimer and Molgaard (1986) mention that glucosinolates and their volatile degradation products can give a positive reaction with alkaline picrate. Mitchell and Richards (1978) describe positive reactions of Brass/ca o/eraceae ssp. o/eracea with the Guignard picrate test. They were not able to detect HCN in these samples Feigl and Anger (1966) report that cyanogen and the dicyanides of sulphur and tellurium also give a positive reaction with their method. In view of the possibility of false positive reactions with species that contain or produce these substances we tested crushed leaves of Brass/ca carnpestr/s, known to produce volatile nitriles and isothiocyanates (Tolstein and Bergstr6m, 1988) and synthetic acetonitrile. In both cases only negative results were obtained. Hegnauer (1986) has drawn attention to the fact that some acyanogenic pJant species produce a different, unstable colouration of the FeigI-Anger test paper. We found a stable brown colouration
521
CYANOGENESIS DETECTION USING MICROTITERPLATES TABLE 1. PLANT SPECIES AND ORGANS TESTED FOR CYANOGENESlSWITH THE MICROTITERPLATEMETHOD Plant species Acacia erio/oba E. Meyer Escholb'a califomica Cham. Lotus corniculatus L. Manihot esculenta Crantz. Prunus laurocerasus L. Prunus lusitanica L. Ranunculus repens L. Sorghum bicolor (L.) Moench Taxus baccata L.
Trifolium repens L. Triglochin maritima L.
Organ leaf leaf leaf flower leaf leaf leaf fruit leaf leaf leaf arillus seed leaf flower leaf
Genotypes tested 2 3 3 3 2 7 1 1 17 2 15 2 2 > 1000 7 25
Cyanogenic compound(s) acacipetalin triglochinin/dhurrin linamarin/Iotaustralin linamarin prunasin amygdalin? triglochinin/dhurrin dhurrin taxiphyllin
linamarin/Iotaustralin triglochinin
Reaction positive positive positive positive positive positive negative positive positive and negative positive positive and negative negative negative positive and negative negative positive and negative
Cyanogenic compounds after Tjon Sie Fat (1979). The presence of acacipetalin in Acacia erioloba has been confirmed by Seigler eta/. (1976).
when testing the cotyledons of cyanogenic and acyanogenic Acacia species (Kakes, unpublished results). The brown colouration is very different from the blue colour produced with HCN. Preparation of the FeigI-Anger testpaper (Tantiwesie et aL, 1969). Freshly make 1% v / w solutions in chloroform of copper (ll)-ethylacetoacetate (Kodak) and di-(4-dimethyl-aminophenyl)-methane (BDH). Mix equal volumes of the two solutions and soak strips of filter paper (Whatman No. 1) in the mixture. Evaporate the chloroform in a fume cupboard. The dry test paper can be kept in a dark, cool and dry place for several months.
References Almazan, A. M. (1987) A guide to the picrate test for cyanide in Cassava leaf. Publ. International Institute of Tropical Agriculture, Ibadan, Nigeria. Brimer, L. (1988) Determination of cyanide and cyanogenic compounds in biological systems. In Cyanide Compounds in Biology (Evered, P. and Harnett, S., eds), p. 177-200. John Wiley, Chichester, U.K. Brimer, L. and Molgaard, P. (1986) Simple densitometric method for estimation of cyanogenic glycosides and cyanohydrins under field conditions. Biochem. SysL Ecol. 14, 97-103. Feigl, F. and Anger, V. (1966) Replacement of benzidine by copper ethylacetoacetate and Tetrabase as spot-test reagent for hydrogen cyanide and cyanogen. Analist91, 282-284. Frehner, M. and Conn, E. E. (1987) The linamarin 13-glucosidase in Costa Rica wild Lima beans (Phaseolus lunatus L.) is apoplastic. Plant Physiol. 84, 1296-1300. Hegnauer, R. (1986) Cyanogene Verbindungen. In Chemotaxonomie derPflanzen VII, 345. Birkh~user, Basel. Kakes, P. (1985) Linamarase and other I{}-glucosidases are present in the cell walls of Trifolium repens L. leaves. Planta 166, 156-160. Kakes, P. (1987) On the polymorphism for cyanogenesis in natural populations of Trifolium repens in the Netherlands I. Distribution of the genes Ac and Li. Acta Bot./Veer/. 36, 59-69. Mitchell, N. D. and Richards, A. J. (1978) Variation in Brassica oleracea L. subsp, oleracea (wild cabbage) detected by the picrate test. N e w Phytol. 81, 189-200. Nahrstedt, A., Erb, H. and Zinsmeister, H. D. (1981) Isolation and structure elucidation of cyanogenic glycosides. In Cyanide in Biology (Vennesland, B. et aL, eds), pp. 461-471. Academic Press, London. Saupe, S. G., Seigler, D. S. and Escalante-Semerana, J. C. (1982) Bacterial contamination as a cause of spurious cyanide tests. Phytochernistry21, 2111-2112. Seigler, D., Dunn, J. E. and Conn, E. E. (1975) Acacipetalin in Acacia constricta from North America. Phytochemistry 15, 219-220. Tantiwesie, B., Ruygrok, H. W. L. and Hegnauer, R. (1969) Die Verbreitung der Blaus~ure bei den Cormophyten. 5. Mitteilung: Uber cyanogene verbindungen bei den Parietales und bei einigen weiteren Sippen. Pharm. Weekblad 104, 1341-1354. Tjon Sie Fat, L. A. (1979) Contribution to the knowledge of cyanogenesis in angiosperms. Doctoral Thesis Leiden. Tollstein, L. and Bergstr6m, G. (1988) Headspace volatiles of whole plants and macerated plant parts of Brassica and Sinapis. Phytochernistry 27, 2073-2077.
522
P. KAKES
Williams, H. J. and Edwards, T. G. (1980) Estimation of cyanide with alkaline picrate. J. Sc~ Food. Agric. 31, 1522. Zitnak, A. (1973) Assay methods for hydrocyanic acid in plant tissues and their application in studies of cyanogenic glycosides in Manihot esculenta. In Chronic Cassava Toxicity (Nestel, B. and Maclntyre, R., eds), pp. 89-96. London.
0305-1978/91 $3.00+0.00 © 1991 Pergamon Press plc.
A Rapid and Sensitive Method to Detect Cyanogenesis Using Microtiterplates P. KAKES Faculty of Biology, Free University of Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Key Word Index--Cyanogenesis; cyanogenic glucosides; ~-glucosidase. Abstract--A method to test plant material for cyanogenesis (the production of HCN) is described. The test requires small amounts of plant material and uses only inexpensive and readily available materials. The method is particularly well suited for field studies. The method has been tested on 11 plant species, in a range of plant tissues.
Introduction Cyanogenesis, or the production of HCN, is a common phenomenon in the plant kingdom. Among the higher plants some 1000 species have been reported as cyanogenic. Very often the evidence rests on the study of a few individuals of one species. Detailed studies on a limited number of species have shown both continuous variation for the amount of HCN produced and polymorphism in the strict sense: the o c c u r r e n c e of cyanogenic and acyanogenic plants within one population. For a better understanding of the o c c u r r e n c e and significance of cyanogenesis it is important to study representative samples of populations, and to study different tissues at different times in the growing season. Although quantitative tests are indispensable, often a semi-quantitative test does give sufficient information. Such tests are generally better suited to study larger numbers of samples. We will describe a method that is quick (100 samples take less than 1 h of preparation time and can be processed within one day), requires small amounts of plant material and uses only readily available materials. Several authors have in the last few years published methods to detect cyanogenesis in plant material [i.e. Brimer and M~lgaard (1986), Brimer (1988), Nahrstedt e t a / . (1981), Almazan (1987)]. Reviews and comparisons of methods have been published by Brimer (1988) and Zitnak (1973); Hegnauer (1986) has discussed the advantages and disadvantages of the FeigI-Anger method (Feigl and Anger, 1966), compared to several other procedures to detect HCN. The method to be described in this paper is different from earlier ones in the following respects. (i) By using a small volume: 0.3 ml for standard microtiterplates--the diffusion time is shortened and the method rendered more sensitive, allowing the use of very small amounts of material. (ii) The use of FeigI-Anger test paper instead of the commonly used alkaline picrate paper has the advantage of a short reaction time. The blue spot is developed almost instantaneously, whereas the alkaline picrate reaction takes 24 h to complete at 28°C (Williams and Edwards, 1980). Short diffusion and reaction times ensure that the rate of colour development is dependent on the rate of HCN formation in the plant material, which is important in enzyme studies. Long incubation times (24 h and longer) are undesirable in view of the spurious positive reaction caused by bacterial contamination of the samples (Saupe eta/., 1982). (iii) The FeigI-Anger test is more specific than the alkaline picrate reaction, reducing the o c c u r r e n c e of false positives. (iv) In field studies the material can be collected directly in the wells of microtiter plates. In that way 96 samples can be transported and processed in a very small (Received 25 November 1990) 519
520
F~ KAKES
volume: 12x8x1.5 cm. (v) The method avoids the use of toluene and picric acid, both potentially hazardous chemicals.
Cyanogenesis and cyanotypes Plants that spontaneously produce HCN when the cells are damaged are called cyanogenic. Cyanogenesis is the result of the hydrolysis of cyanoglucosides by specific J3-glucosidases. It is presumed that cyanoglucosides are present in the vacuole; the corresponding 13-glucosidases have been shown to be located in the cell walls in two species: Phaseolus lunatus and Trifolium repens (Frehner and Conn, 1987; Kakes, 1985). In species monomorphic for cyanogenesis the plant usually contains both cyanoglucoside(s) and specific 13-glucosidase(s). In polymorphic species the acyanogenic genotypes or organs may lack the cyanoglucosides, the corresponding ~-glucosidase or both. In such cases, four cyanotypes may be distinguished by adding cyanoglucosides or a j]-glucosidase to the sample. If the nature of the cyanoglucoside(s) in the species is not known, it may be useful to add a broad spectrum j]-glucosidase to (part of) the sample. Helicase, a mixture of enzymes from the gut of Helixpomatia, may be used in this case. Cyanolipids are not hydrolysed by helicase, and some cyanoglucosides very slowly (Brimer, 1988) so samples that give negative results need further examination. Description of the Method The samples are placed on the bottom of the wells of a microtiterplate. Wells of 300 ~1 are suitable for leaves of Tr/fo//um repens and Lotus cornicu/atus, larger wells may be used for more voluminous or weakly cyanogenic samples. The microtiterplates are placed in a refrigerator at -20°C, for at least 1 h, After thawing, 100 p.I of distilled water is pipet-ted in each well, the surface of the plate is carefully wiped dry and a sheet of FeigI-Anger test paper (see later) is placed over the wells. The sheet is covered with a glass plate of the size of the microtiterplate and the complete sandwich is secured with paperclamps. The sandwich is placed in an incubator at 35°C. After 2-3 h, depending on the material, the paper is removed. Over wells with cyanogenic material blue spots will have formed. The minimum amount of HCN detected is approximately 40 nmol HCN per well. Up to 180 nmol HCN there is a clear relation between the amount of HCN and the intensity of the blue spot. Using leaf disks of a cyanogenic plant of Trifo/iurn repens, I found that 2 mg fresh weight was enough to obtain a faint blue spot. In Trifo//umrepens all four cyanotypes occur. They are easily recognized by complementing the leaf material with linamarin, one of the cyanogenic glucosides in Trifo//urn repens, and with linamarase, the corresponding ~-glucosidase. In this study plants of known genotype were used to test the method. Plants with linamarin, but lacking linamarase gave a clear positive reaction with 10 mU of linmarase, plants lacking linamarin but containing linamarase gave a positive reaction with 40 nmol of linamarin. Linamarin and linamarase may be purchased but relatively crude preparations of both substances give equally good results. Simple methods to prepare linamarin from flax seedlings and linamarase from linseed are described by Kakes (1987). By using known amounts of plant material (i.e. leaf disks) and comparing the spots with a graded series of HCN, the method, essentially qualitative, can be made semi-quantitative. The spots retain colour and intensity for several weeks if kept cool and in the dark. Testing of the method with different cyanogen/c substrates and testing for fa/se positive reactions. Table 1 shows cyanogenic species known to be cyanogenic from the literature, tested with our method. Different plant organs, both cyanogenic and acyanogenic are included. It is clear that the method can be used on a wide variety of species and has good discriminating power. The negative reaction of the leaves of Prunus/us/tan/ca is unexpected, as Rosenthaler (1919) found all parts of this plant cyanogenic. The polymorphism found in Trig/ochin rnar/tirna has to my knowledge not been reported earlier. As small amounts of material may be used, plant parts as leaflets, cut disks of leaves and small seeds may be used, thereby revealing individual and within-plant variation. Brimer and Molgaard (1986) mention that glucosinolates and their volatile degradation products can give a positive reaction with alkaline picrate. Mitchell and Richards (1978) describe positive reactions of Brass/ca o/eraceae ssp. o/eracea with the Guignard picrate test. They were not able to detect HCN in these samples Feigl and Anger (1966) report that cyanogen and the dicyanides of sulphur and tellurium also give a positive reaction with their method. In view of the possibility of false positive reactions with species that contain or produce these substances we tested crushed leaves of Brass/ca carnpestr/s, known to produce volatile nitriles and isothiocyanates (Tolstein and Bergstr6m, 1988) and synthetic acetonitrile. In both cases only negative results were obtained. Hegnauer (1986) has drawn attention to the fact that some acyanogenic pJant species produce a different, unstable colouration of the FeigI-Anger test paper. We found a stable brown colouration
521
CYANOGENESIS DETECTION USING MICROTITERPLATES TABLE 1. PLANT SPECIES AND ORGANS TESTED FOR CYANOGENESlSWITH THE MICROTITERPLATEMETHOD Plant species Acacia erio/oba E. Meyer Escholb'a califomica Cham. Lotus corniculatus L. Manihot esculenta Crantz. Prunus laurocerasus L. Prunus lusitanica L. Ranunculus repens L. Sorghum bicolor (L.) Moench Taxus baccata L.
Trifolium repens L. Triglochin maritima L.
Organ leaf leaf leaf flower leaf leaf leaf fruit leaf leaf leaf arillus seed leaf flower leaf
Genotypes tested 2 3 3 3 2 7 1 1 17 2 15 2 2 > 1000 7 25
Cyanogenic compound(s) acacipetalin triglochinin/dhurrin linamarin/Iotaustralin linamarin prunasin amygdalin? triglochinin/dhurrin dhurrin taxiphyllin
linamarin/Iotaustralin triglochinin
Reaction positive positive positive positive positive positive negative positive positive and negative positive positive and negative negative negative positive and negative negative positive and negative
Cyanogenic compounds after Tjon Sie Fat (1979). The presence of acacipetalin in Acacia erioloba has been confirmed by Seigler eta/. (1976).
when testing the cotyledons of cyanogenic and acyanogenic Acacia species (Kakes, unpublished results). The brown colouration is very different from the blue colour produced with HCN. Preparation of the FeigI-Anger testpaper (Tantiwesie et aL, 1969). Freshly make 1% v / w solutions in chloroform of copper (ll)-ethylacetoacetate (Kodak) and di-(4-dimethyl-aminophenyl)-methane (BDH). Mix equal volumes of the two solutions and soak strips of filter paper (Whatman No. 1) in the mixture. Evaporate the chloroform in a fume cupboard. The dry test paper can be kept in a dark, cool and dry place for several months.
References Almazan, A. M. (1987) A guide to the picrate test for cyanide in Cassava leaf. Publ. International Institute of Tropical Agriculture, Ibadan, Nigeria. Brimer, L. (1988) Determination of cyanide and cyanogenic compounds in biological systems. In Cyanide Compounds in Biology (Evered, P. and Harnett, S., eds), p. 177-200. John Wiley, Chichester, U.K. Brimer, L. and Molgaard, P. (1986) Simple densitometric method for estimation of cyanogenic glycosides and cyanohydrins under field conditions. Biochem. SysL Ecol. 14, 97-103. Feigl, F. and Anger, V. (1966) Replacement of benzidine by copper ethylacetoacetate and Tetrabase as spot-test reagent for hydrogen cyanide and cyanogen. Analist91, 282-284. Frehner, M. and Conn, E. E. (1987) The linamarin 13-glucosidase in Costa Rica wild Lima beans (Phaseolus lunatus L.) is apoplastic. Plant Physiol. 84, 1296-1300. Hegnauer, R. (1986) Cyanogene Verbindungen. In Chemotaxonomie derPflanzen VII, 345. Birkh~user, Basel. Kakes, P. (1985) Linamarase and other I{}-glucosidases are present in the cell walls of Trifolium repens L. leaves. Planta 166, 156-160. Kakes, P. (1987) On the polymorphism for cyanogenesis in natural populations of Trifolium repens in the Netherlands I. Distribution of the genes Ac and Li. Acta Bot./Veer/. 36, 59-69. Mitchell, N. D. and Richards, A. J. (1978) Variation in Brassica oleracea L. subsp, oleracea (wild cabbage) detected by the picrate test. N e w Phytol. 81, 189-200. Nahrstedt, A., Erb, H. and Zinsmeister, H. D. (1981) Isolation and structure elucidation of cyanogenic glycosides. In Cyanide in Biology (Vennesland, B. et aL, eds), pp. 461-471. Academic Press, London. Saupe, S. G., Seigler, D. S. and Escalante-Semerana, J. C. (1982) Bacterial contamination as a cause of spurious cyanide tests. Phytochernistry21, 2111-2112. Seigler, D., Dunn, J. E. and Conn, E. E. (1975) Acacipetalin in Acacia constricta from North America. Phytochemistry 15, 219-220. Tantiwesie, B., Ruygrok, H. W. L. and Hegnauer, R. (1969) Die Verbreitung der Blaus~ure bei den Cormophyten. 5. Mitteilung: Uber cyanogene verbindungen bei den Parietales und bei einigen weiteren Sippen. Pharm. Weekblad 104, 1341-1354. Tjon Sie Fat, L. A. (1979) Contribution to the knowledge of cyanogenesis in angiosperms. Doctoral Thesis Leiden. Tollstein, L. and Bergstr6m, G. (1988) Headspace volatiles of whole plants and macerated plant parts of Brassica and Sinapis. Phytochernistry 27, 2073-2077.
522
P. KAKES
Williams, H. J. and Edwards, T. G. (1980) Estimation of cyanide with alkaline picrate. J. Sc~ Food. Agric. 31, 1522. Zitnak, A. (1973) Assay methods for hydrocyanic acid in plant tissues and their application in studies of cyanogenic glycosides in Manihot esculenta. In Chronic Cassava Toxicity (Nestel, B. and Maclntyre, R., eds), pp. 89-96. London.