\ IT}! M, HI" l“ W W \ w \ \t. W l e -7 , _._,_ :f._ ‘—. — , ,_ ‘\ \ W \ I W MW FACTORS AFFECTING THE HYDROCYANIC ACID CONTENT OF WILD WHITE CLOVER. Trifolium rcpens L.. Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Basil J. Finn 1942 THESTS \{Illll'linl u}f!|l."| (I'llv'H‘..|x,n \‘ \Il'!‘|l\l|ll‘l.a.l’l ‘ \ \f‘ lllIl-tllllll'lfll . .ll 1|‘|I\l' III F»: \‘T‘ FACTORS AFFETING THE HYDROCYANIC ACID CONTENT or WILD WHITE CLOVER. Trirolium repens L. FACTORS AFFECTING m HYDROCYANIC ACID CONTENT 01' WILD WHITE CLOVER; Trifolium ‘repens L. , A Thesis Respectfully Submitted in Partial Fulfillment of the Requirements for the Degree or Master of Science at Michigan State College of Agriculture and Applied Science. hall I . Finn 1948““ wears ' C 0 N T E N T S INTRODUCTION 0......0.0.0.0....0OOOOOOOOOOOOOOOOOOOOOOO REVIEWOF LIMATURE OOOOOOOOOOOOOOO0.00.000.000.000... STUDIES CONTENT GENERAL HON Content of Various Crops .................. Toxicity of Cyanide ........................... Lethal Dose ................................... HCN Content in White Clovers .................. Factors Affecting the HON Content ............. Climate and Soil ......................... Effect of Fertilizers .................... Diurnal and Seasonal variations .......... Influence of Frost ....................... Different Stages of Growth Affect the HCN Content .................................. Inheritance of Cyanogenesis ................... 0N ‘IHE INCLUENCE OF CERTAIN FACTORS ON THE HON OF WILD WHITE CLOVER The Influence of Soil Mbisture on the HON Con- tent of White Clover (Experiment 1) ........... The Influence of Season on the HON Content of White Clover (Experiment 2) ................... The Influence of Temperature on the HON Content of White Clover (Experiment 3) ................ The Influence of Light on the HON Content of 'White Clover (Experiment 4) ................... Correlation oleCN'Content with Habit of Growth (Experiment 5) ................................ DISCUSSION 0.00.0.0...0.00.00.00.000.0.0.000... 143383 Page GN‘IQOIFIFNNH +4 ta cs re 13 14 16 25 29 33 37 39 Page SUMMARY ................................................ 42 ACKNOWLEDGMENTS ........................................ 42 REFERENCES ............................................. 43 APPENDIX ............................................... 46 (Tables I to VII) FACTORS AFFECTING THE HYDROCYANIC non) CONTENT or WILD WHITE CLOVER (Trifolium repens, 1,.) INTRODUCTION It has been well established that a large number of plant species contain quantities of hydrocyanic acid which apparently is in the form.of some non-toxic compound of cyanide, since it is well known.that free cyanide is strongly toxic to plants. This compound may be one of several glucosides which under certain conditions, such as the crushing of the plant, break down under the action of associated enzymes into hydrocyanie acid and other pro- ducts. In the white clover breeding program.carried on by the Division of Forage Plants, Central Experimental ‘rarm, Ottawa, material collected from.the vicinity of thpan, N.S., where the species is thought to be indigenous or highly adapted, has been used extensively and has prov- ed to be so promising that it has been studied from several angles. One of these lines of approach was to study some of the factors which affect the variability of the hydrocyanic acid content of this wild white clever and to consider the importance that the presence or ab- sence of hydrocyanic acid content might have in.the breed- ing pregram. The results of this study are presented in this paper and, while no very definite conclusions can be drawn from these results, they may be of value to the white clover breeder. -3- REVIEW OF LITERATURE The review of literature set forth herein includes some of the articles on.factors affecting the HCN'content in grasses and wild white clover. As early as 1906 Dunstan and Henry (8) stated that HON was found in more than 100 different plants belonging to 22 different natural orders. In the same year Greshoff (12) published a very complete list of plants in which cyanogenetic glucosides were traced, but unfortunately little information is available in his papers concerning the amounts of the glucosides present in the plants. Except in the case of sorghum, Sudan, and Johnson grasses, there are very little data on the amounts of HON in plants. _H_C_N Content of Various Crops Numerous workers have used various quantitative methods in calculating the EC! content of different forage crops and they have expressed their results in different ways. For the purpose of comparing the results of these workers, their findings have been converted to parts per million (p.p.m.) on the green basis, as shown in Text Table I. The data in Text Table I indicate that there is a wide variation between crops (compare the HON content of Johnson grass and brown top). There is also a wide variation shown within crops (compare the different amounts of RCN' found in sorghum or in wild white clover). -3- Text Table I Comparative Concentration of HON in.Various Crops (expressed in p.p.m. on the green basis) Author Crop p.p.m. Remarks Dowell (7) Johnson grass 5740 ithimum.obtained Iranzke et a1 (11) sorghum. 2540 do (3 year average) Willamen & West (38) do 1140 Maximum obtained Franzke et a1 (11) do 410 do do (11) Sudan grass 420 As check plant, not min. obtained Swanson (35) do 150 Maximum obtained Miranda (19) wild w. clover 36-390 Book (6) do 10-130 Sullivan (34) do 10-320 Greenhouse test do (34) do 500 very high content (one plant only) TRigg et a1 (29) do 16-124 Eleven samples from different countries Tinn do 28 do (29) alfalfa 13 do (29) red clover 3 do (29) alsike 3 do (29) fescues 2 do (29) brown top 1 * D.p.m. on the Green 0 "' 50 e 50 " 100 e 100 - 150 . 150 - 200 . :aZOO . * Assuming that dry weight of Sudan grass is 20$ Basis ‘Relative Degree of Toxicity .Very low (safe to pasture) .Low (safe to pasture) .Medium (doubtful) .High (dangerous to pasture) .Very high (very dangerous to pastzure) (After Boyd et a1 (3)) -Toxicitz of Cyanide Leeman (14) indicates that various species of animals react differently when fed plants containing cya- nophoric glucosides. These differences are caused by dif- ferent anatomical structures and different detoxifying a- bilities of various animals. Cattle and sheep are ruminants and both are known to be subject to poisoning by cyanophoric glucosides. The paunch, or rumen, of these animals is neither strongly acid nor alkaline in reaction and contains a large flora of micro-organisms and considerable quantities of the enzyme emulsion which.provide an excellent medium.for the liberation of the toxic agent. 0n the other hand, horses and hogs, being non-ruminants, have only one stomach which is strongly acid due to the presence cf.hCL which inhibits the release of HON. Lethal,Dose There are comparatively few experiments in grasses which have been properly conducted with a view to estimating their toxicity; ‘Hindmarsh (13) found, in administering Scheele's acid (HON), that the lethal dose per 1 pound body weight of sheep or cattle was 1 mg. Seddon and King (33) confirmed.Hindmarsh's determination of 1 mg. per pound body weight for sheep. Retrie (23) reported on.an experiment made with ngodon incompletus (a grass indigenous to Aus- tralia). This grass containing 160 p.p.m. cf prussie acid was fed to sheep. It was estimated that the lethal dose per sheep of 150 pounds was two pounds of grass, capable of -5- liberating 0.14 grams of prussic acid. This confirmed again the figure established by Hindmarsh (13) as, roughly, 1 mg. per pound body weight. Leeman (14) points out that the important question is not how much HCN is introduced but how much of it can be liberated. One way of solving the question.wcu1d be to deter- mine the prussic acid content, first before it was fed, and then after the death of the animal. The grass found in the paunch could be retested. Another point to be considered is that of elimination from the animal body. Prussic acid is highly diffusible and will readily reach the blood stream“ but it will by this same prcperty be eliminated very quickly. The difference between the quantity of EUR eliminated and that contained in the ingested material will determine, to a large degree, the toxicity of the dose. The condition of the animal prior to eating the toxic plant must be taken into account as certain factors, such as hunger, overstrain, bad health, and thirst, exert considerable influence on the biological reactions. An animal that is in.poor health or extremely humgry has been found to be more susceptible to the poison. There have been occasional references where cattle were poisoned by substances other than.HCN. As early as 1897 Pease (22) showed that mortality of cattle in India from.Johnson grass could be imputed to nitrate poisoning. He was able to detect 20 per cent of potassium nitrate in stems of the grass. A.very recent case occurred at South -5- Dakota State College, when.Dr. Lipp, Staff veterinarian, was called out to diagnose the case of 20 young cattle that had died after eating Sudan grass which had been stored in a stack. On analysis the Sudan forage was found to contain no appreciable quantity of HCN but tests revealed that it con- tained 8.47 per cent of nitrates. ggN’Ccntent in White Clover; Miranda (l9) and Armstrong et a1 (1) observed that individual plants of white clover fall into two physiological groups : one in which the leaves produce minute quantities of prussic acid when they are killed, and the other in which this phenomenon does not occur. Armstrong et a1 (1) drew attention to the fact that, in their experience, the wild plant wherever tested always contained cyanide and that they had not succeeded in finding cyanide in white clover raised from "cultivated" seed at any stage of growth. From.this it appears possible that the property of cyanOgenesis might in some way be connected directly with the wild state and that its absence might be correlated with the cultivat- ed condition of the plant. The results obtained by Pethy- bridge (25) showed however that, inter alia, seedlings from some samples of commercial ”cultivated" seed gave a positive reaction when tested forgHCN. ZHe found that in testing 126 samples of commercial white clover 109 gave negative results and only 17 positive reactions. But of these 17 samples which were cyanophoric 15 were of United States and -7- ‘2 of Canadian origin. He also conducted numerous experiments with commercial English white clover, and it must be said that all his tests with these seedlings were invariably negative. Factors Affecting the HON Content The literature on environmental factors is very extensive, including many conflicting statements and theories. Some of the various conditions which affect the prussic acid content in different crops are stages of maturity, rainfall, drought, frost, insect damage, moulds or fungi, fertilizers, temperature, and humidity. In this review of literature only a few of the factors which affect the HON content in crops will be discussed. Climate and Soil:- Miranda (19) found as early as 1912 that the character of the soil influenced the HON content in wild white clover. In their study of sorghum, Willaman and west (39) concluded that climate was more influential than soil on the prussic acid content. They reported that an adequate water supply is usually accompanied by a low content and an inadequate supply by a high amount of prussic acid. ‘ Franzke et a1 (11) demonstrated that increases in soil moisture showed increased growth and gradually decreased.HCN in sorghum, as shown in Text Table II. -8- Text Table 11 RON Content in Two Strains of Sorghum.Grown. in Greenhouse Cultures with varying Mbisture Contents (1938) Moisture ng HCN train High HCN Strain per cent ‘Ave. weekly' HCN .Ave. weekly HCN Growth p.p.m. Growth p.p.m, Inches Inches 15 1.417 880 1.417 3920 20 1.500 750 2.458 2120 25 2.583 510 2.417 2270 30 3.125 590 2.917 1570 35 3.167 380 3.125 1410 From.the foregoing it appears that the amount of HON decreased with the same regularity as the weekly growth in inches increased together with the regular increase of water added to the soil. In studies of the same plant, Peters et a1 (24) stated that growth arrested by drought presented a very favourable condition for the elaboration of the poison, which observation was also confirmed by the findings of Francis (10). Vinall (36), after a critical survey of litera- ture, concluded that injury to growing sorghum.p1ants by drought increased the HON content, but that stunted growth from lack of plant food in the soil diminished it. On the contrary, Rogers and Boyd (30) have shown in their investiga- tions of Sudan grass that plants exposed to drought conditions contained less HON than plants grown under normal conditions. -9- Effect gf Fertilizers:- , Several workers have studied the effect of nitrogen on the HON content of sorghums. Maxwell (16) pointed out that soil rich in nitrogenous constitutents of plant food attended higher prussic acid. In the same year Brannick (4) concluded that soils,to which sodium.ni- trate had been added, produced plants which contained slightly more HON than the unfertilized plants. The same worker also applied sodium nitrate to millet and found that it behaved similarly. Willaman and West (39) discover- ed that nitrogen added to a poor soil with abundant nitrogen will not show any effect after fertilization. On the other hand, Manges (15) of Kansas in 1936 stated: "Plants grown on fertilized soils, especially soils which have been far- tilized with nitrates, contain less HCN'than those grown on poor soils?, indicating disagreement with previous workers. Boyd et a1 (3), in their recent ‘work on Sudan grass, Observed that a high level of avail- able nitrogen and a low level of available phosphorus in the soil tend to increase the poison.content, while a low level of available nitrogen and a high level of available phosphorus have the cpposite effect. They reported also that potash had no influence on the HON content. In the following year, Franzke and co-wcrkers (11) made a similar study of fertilizer tests on the HCN'content of sorghums. The kind of fertilizers regularly applied and the amount of'HCN found in the samples \ -10- .from the four strains studied are given Text Table III. Text Table III HCN Content (p.p.m,) in Plants of Four Selfed Strains Grown on Complete Fertilizer Plots in Brookings (1937) Strain None 11.330 P'200 K'200 H.350 N'350 P200 H.350 P'ZOO P200 I'200 P200 £3200 1-30-I 3610 3860 4130 2720 '4660 3380 2770 4430 15-30-8 5200 6220 5870 3810 6740 5890 3660 5580 18-30-8 4900 -- 4110 3960 5690 4970 4310 5400 39-30-8 2230 2980 2030 1960 2090 2930 1380 2320 Average 3985 4353 4035 3113 4795 4293 3030 4433 Insight 13.9 10.7 12.5 16.0 14.8 11.6 15.5 13.6 From the above table, the average amount of HON was invariably higher in plants where nitrogen was regularly applied than where nothing was applied. It was likewise true that the HON content was higher in all cases where nitrogen was used with superphosphate than where super- phosphate was used alone. Moreovar, the corresponding amounts of HCN'were higher where nitrogen was added to both phosphorus and potassium, than where the latter were applied together without nitrogen. From the foregoing it would appear that, for some reason, the regular use of nitrogen in the cropping system.was correlated with an increase in the amount of prussic acid. -11.. The HCN content of plants on potassium.plots was invariably lower than that of un- fertilized Specimens. Leeman (14) summarized the influence of soil on the HON content as follows: "Although it is thus demonstrated that the soil has a marked effect on the prussic acid production, which is interesting from.an academic point of view, from a practical point of view the increase is not such as to warrant any further investigations in that direction." Diurnal and Seasonal variations:- The production of prussic acid due to diurnal variations has frequently been reported. Revenue (28) observed that there was an increase in.HCN in sorghum from early morning to afternoon. Willaman and West (39) in studies of the same plant also noticed a gradual in- crease from morning to mid-day, at which time the HCN content was at its maximum. In the case of Sorghum vul- gggg,iNarasimha.Acharya (20) found that the HCN’increasad from early morning to about 2 p.m,, after which time there was a slight decline until 6 p.m., followed by‘a rapid decline at night. He was of the opinion that there was a correlation between.HCN’and photo-synthesis. Marias and Rimingtcn (17), in their study of Dimorphotheca cuneata, Less., found that the prussic acid increased from early morning until noon, which they thought "suggests a correlation with intense (l) -12- photosynthetic activity". The more recent work of Boyd at al (3) showed definite diurnal variations in the cyanide con- tent of Sudan grass and sorghum, Samples were taken for analysis at 8.00 a.m,, 1.00 p.m,, and 7.00 p.m. In the case of Sudan grass, the HCN content at 1.00 p.m. was about 30% higher than the samples taken in the morning and evening. In the case of sorghums, the cyanide content at 1.00 p.m. was also considerably higher than in the morning and evening. It is to be noted that these results are in accordance with those reported by Narasimha Acharya (20). The data obtained in this experiment were subjected to statistical analysis, and the differences between the cyanide content at noon, morning and evening were found to be significant. In sum- marizing the diurnal effect, it may be concluded that the foregoing workers were in fairly close agreement. In addition to diurnal changes, there is a pronounced seasonal variation in the HCN content of different crops. In 1933, Askew (2) in his studies of wild white clover discovered a seasonal variation. Again in 1937 Rogers and Frykolm.(31) noted that the percentage of white clover plants containing no HCN decreased from 71.28 to 57.60 with the advance of season. ' Ramsay and Henry (27), in their study of whitewood (Heterodandron oleaefolium) and native fuschsia (Ergmphila maoulata), reported that the HCN content reached a maximum.during late summer. -13- Influence of Frost:- Frost, - according to Peters, Slade, and Avery (24), did not influence the HCN content of sorghum except as a forerunner of a pe- riod of bright dry weather. In such a case, the bright warm weather was conducive to the growth of new tillers which are always higher in.HCN content than the more mature parts of the plant. Manges (15) for the same reason, in the study of Sudan grass, stated that the HON content increases after the first frost. In experimenting with the same crop Rogers and Boyd (30) observed an increase after frost, but they did not record any deaths imputable to this factor. Vinall (36) found that the prussic acid content of sorghum increased after frost, whereas Francis (10) stated merely that frosted sorghum.plants were unsafe to pasture. The.more recent work of Boyd et a1 (3) showed that in the case of Sudan grass the HCN content was no higher after frost than before it. Different Stages of Growth Affect the HCN'Content:- Narasimha Acharya (20) pointed out that in a normal crop of Sorghum vulgare the prussic acid decreased from the early stages progressively to the flowering stage. Previous workers, Peters et a1 (24), Willaman and west (39), Manaul and Dowell (18), also found a decrease from early to later stages of growth. Dunstan and.Henry (8) observed that the stage at which maximum.HCN prevailed varied considerably with dif- .ferent plants. Williams (40) found that the HON content of wild white clover decreased -14- rapidly with the age of the leaves as shown in Text Table Iv. Text Table IV" HCN Content of Wild White Clover Leaves at Different Ages Plant No. Ybung folded Few days old Few weeks old leaves 1 Very strong medium very weak 2 Strong ‘Medium. weak 3 very strong Very weak None 4 very strong Strong weak Inheritance of Cyanogenesis The recent work of‘Williams (40) relating to the genetics of cyanogenesis in white clover (Trifolium.repens) showed that the extreme variations in the HON content of young leaves of different plants were due less to differences in environmental condition than to some intrinsic property of the individual. The cyanogenetic reactions of the plants were tested by Guignard's picrate paper method described by Armstrong at al (1). Crosses made between homozygous HCN'plants and homozygous free HCN plants gave evidence of dominance of positive plants to negative plants. Segregation for cyano- genesis in backcrosses resulted in a 1 : 1 ratio expected on the basis of simple Mendelian segregation. The segregating generations resulting from.heterozygous crosses showed a -15- 3 : 1 ratio of positive HCN plants to negative HCN plants. Sullivan and cosworkers (34) conducted similar ex- periments on the inheritance of cyanogenesis in wild white clover. These American workers confirmed the findings of Williams (40), namely, that cyanophoric plants were dominant to acyanophoric plants. Recently Franske et a1 (11) investigated the in- heritance of prussic acid in sorghum. They reported that acyanOphoric plants appeared to be partially dominant to cyanophoric plants. According to Coleman and Robertson (5) of Colorado State College, the differential ability of inbred lines of Sudan grass to produce HCN may be inherited. In their tests, high HCN’production appeared to be more closely associated with non-glossy leaves than with glossy leaves. Similarly, high.HCN content was concomitant with purple-tinged seedling leaves, but to a lesser degree. -16.. - STUDIES ON THE INFLUENCE OF CERTAIN FACTORS - ON THE HCN CONTENT OF WHITE CLOVER The Influence of Soil Mbistura on the HCN'Content of White Clover (Experiment 1) Experimental Procedure This experiment was set up in the greenhouse during the summer of 1939 in an effort to ascer- tain the effect of the quantity of soil moisture on the HCN content of white clover. Material used:- After a preliminary test of a fairly large number of plants, three groups of 5 plants each were selected on the basis of their HCN content. One group was classified as having high HCN content, another medium HCN content and the other low HCN content. Each individual of the three groups was then divided vegetatively into nine separate units. These were started in 3-inch clay pots con- taining good loam soil wnich was thoroughly mixed to ensure uniformity of growth. This made it possible to have avail- able, for each of the three treatments used, 3 units of each of the five plants in all three groups. Treatments:- The treatments meant that a certain percentage of soil moisture had to be maintained in the pots, as follows: Treatment 1 - 34 per cent soil moisture 2.22:: n w w 3 - 15 ” " ” ” -17 This was accomplished by weighing the pots daily and bringing them up, by watering, to the pre- determined weight required for such a percentage moisture. Method of Tasting:- .A review of literature snows that numerous quantitative methods have been used in estimating the HCN content of various crops. These methods require a considerable amount of time and the use of fairly large quantities of material. Since numerous individual units of wild white clover had to be studied in this experiment, a rapid method adaptable to small quantities was required. The method used is that of Nowosad and MacVicar (21) which is an adaptation of the picric-acid method previously used by such workers as Pethybridge (25), Sampson (32), Foy and.Hyde (9) in studying the HCN'content of white clover. This method is based on the evolu- tion of hydrocyanic acid and the reaction of this acid when it comes in contact with filter paper treated with an alka- line picrate solution. The details of the preparation of filter paper and standards for colorimetric readings were carried out as outlined by Nowosad and macVicar (21). By using a Duboscq colorimeter, exact quantitative readings were made which were later calculated and expressed in.Mg. HCN per c.c. of solution. In sampling, two samples of five compound leaves were taken from.aach unit, from.which the average HCN content was calculated. The sizes of the leaves -18- were found to vary slightly under different treatments. To_ offset any error due to a difference in leaf size, precaution was taken to sample only those leaves which were of average size. Young folded leaves were avoided in sampling as they have been found by Williams (40) to be higher in.HCN content than mature leaves. Diseased and injured leaves were also avoided. All samples were made at approximately the same time of day (early afternoon). This precaution was taken because of the possibility of diurnal influence as mentioned in (28), (39), (20), (17), (3), (2), (31). Sampling of all units was carried out at lO-day intervals over a period of 70 days. For the purpose of comparing the HCN content of wild white clover with the findings of other workers, the quantitative method of Boyd at al (3) was used to test one plant of moderate HCN content. It was found to contain 28 p.p.m, of HCN on the green basis (see Text Table I). Results:- While, as was expected, considerable variation was found when the data were analyzed, it was possible to subject the findings to statistical analysis and to draw conclusions on the basis of such analyses. The following tables indicate the influence of the soil moisture treatments on the HCN content of each of the three plant groups under study. -19- -Text Table V Analysis of variance for Treatments of Plants "High" in.HCN Content at the End of 40 Days. (For complete data see Appendix Table I.) Due to n.r. M.S. 1' Value 3 Value “1‘ ‘5p.c. lgp.c. Treatments 2 .00123060 49.98 3.26 5.25 Plants 4 .00003080 1.23 2.63 3.89 Replicates 2 .00001560 Error 36 .00002460 Treatment Means No. MOisture Level “3°‘HCN/°'°° l 34 p.c. soil moisture .0449 2 22 p.c. soil moisture .0386 3 15 p.c. soil moisture .0270 Necessary difference for significance (P = .05) .0035 (P’- .01) .0047 For (P=.05) treatment 1 is significant to treatments 2 and 3, and " 2 " " " treatment 3. For (P=.01) treatment 1 is highly significant to treatments 2 and 3, and " 2 " ' * ' treatment 3. It will be observed that the'high" lHCN'plants reacted significantly to the different moisture levels. Treatment No. 1, in Which the moisture level was main- tained at 34 per cent, produced a mean quantity of .0449 mga -20- per c.c., which was significantly higher than the .0386 mg.. of treatment 2 (22% moisture) and the .0270 mg. of treatment 5 (15% moisture). Furthermore, treatment 2 gave significant results over treatment 3. Text Table VI Analysis of variance for Treatmenhaof Plants "Medium" in HCN Content at the End of 40 Days. (For complete date see Appendix Table II.) Due to 13.3. M.S. F Value F Value for 5 p.c. 1 p.c. Treatments 2 .000125 125.00 3.26 5.25 Plants 4 .000678 678.00 2.63 3.89 Replicates 2 .000004 4.00 3.26 5.25 Error 36 .000001 Treatment means No. Mbisture Level mg. HCN/c.c. l 34 p.c. soil moisture .0299 2 22 p.c. " ” .0277 3 15 p.c. " " .0241 Necessary difference for significance (P = .05) .0007 (P = .01) .0010 For (P = .05) treatment 1 is significant to treatments 2 and 3, and 7 2 " " " treatment 3. For (P = .01) treatment 1 is highly significant to treatments 2 . and 3, and " 2 " " ” ” treatment 3. -31- A perusal of this table shows the . reaction of the group "medium” HCN to the moisture levels, in- dicating results very similar to those obtained with the "high" group. While the mean differences are much smaller, ranging from .0299 mg. for treatment 1 to .0241 mg. for treat- ment 3, the necessary difference for significance is also much lower. Text Table VII .Analysis of variance for Treatmentsof Plants ”low" in HCN Content at the End of 40 Days. (For complete date see Appendix Table III.) Due to D.F. M.s. F value F value for 5 2.0. lpeco Treatments 2 .00004808 6.74 3.28 5.25 Plants 4 .00057125 80.12 2.63 .3.89 Replicates 2 .00000109 Error 36 .00000713 Treatment means No. MOisture Level . mg. HCN/c.c. 1 34 p.c. soil moisture .0163 2 22 p.c. " " .0158 3 15 p.c. " ” .0130 ' Necessary difference for significance (P = ‘05) 00019 (P = .01) .0025 .For (P = .05) treatments 1 and 2 are significant to treatment 3. IFor (P = .01) treatments 1 " 2 " highly significant to treatment 3. ’ -33- While the reaction of the "low"- HCN'group was along the same lines as that of the "high" and ”medium? groups with.HCN content ranging from .0163 mg. to .0130 mg., it will be observed that treatment 1 did not show significance over treatment 2. It is probable that the "low" HCN’content of this group accounted for varia- tions so small that they could not be detected. Text Table VIII Analysis of Variance for Soil maieture Treatments Applied to Plants of three HCN Levels, i.e. "hi ”, "low” HCN Content. see Appendix Table IV.) "mediumfl, and For complete data Due to DJ. 11.8. P Value 1' “1“" “1' 5;2.c. 1 .c. Treatments 2 .00093902 192.42 3.13 4.92 men content 2 .00538142 1102.75 3.13 4.92 Replicates 18 .00000724 1.48 1.75 2.21 Plants 12 .00042687 87.47 1.89 2.45 Treatments 1 man“ 24 .00003092 5.34 1.67 2.07 fTreatments x m Content 4 .00023271 47.69 2.50 3.50 lrror 72 .00000488 (Continued) -33- Treatment Means Ho. Moisture Level Mg. HCN/c.c. l 34 p.c. soil moisture .0304 28 p.c. I. Q .0273 i 3 15 p.c. " " .0214 necessary difference for significance (P = .05) .0009 (P = .01) .0012 For (P = .05) treatment 1 is significant to treatments 2 and 3, and " 2 " " " treatment 3. For (P = .01) treatment 1 is highly significant to treatments 2 and 3, and " 2 " " " " treatment 3. _ _ A combined analysis of the data for the three groups showed that treatment 1 with .0304 mg. HCN was significantly higher than treatmentsz and 3 with .0273 mg. and .0241 mg. respectively. Treatment 2 was also significantly higher than treatment 3. The data obtained have been plotted graphically in fig. 1. This graph shows the HON content over a 70-day period for each of the three treatments. Discussion:- The obvious conclusion that must be drawn from the results of this experiment is that an adequate supply of soil moisture is followed by a high concentration of HCN, while a restricted moisture supply is associated with a. A.®peowfim5© me ecpmop was meEHP monzp Umpwowfigmh .mwmdfim m mo GeeE exp mp topsomenemn we psefipeohp_£osmv .vmmp newton a Leeo peepcoo 20m exp :0 mpemfipseep enepwfloa Hflom means mo DoseSHmee Dee .H .mfle 2%: 3} {mm QB a\m Prom <8. {2 mmo. m escapee-Hm. ( . I N Puma-#895 I l I e\. /e - .i\\\ Illl:/, H peofipseae / 80. 7 / / mmoe / / 0/ e \ -I I 1‘) I, u 060. mwo. . uni-Sm . we _ . -24- lower concentration. Rogers and Boyd (30) report similar findings in their study of Sudan grass when they observed that plants exposed to drought conditions contained less HCN than those grown under normal conditions. On the other hand, ‘Willanan and west (39), while working with sorghum, concluded than an adequate supply of soil moisture is usually accompanied by a low concentration of HCN and that an inadequate supply of soil moisture is usually accompanied by a high concentration of‘HCN; 'Likewise, in studies of the same crop, Franske et a1 (11) demonstrated that increases in soil moisture showed increased growth attended with a reduction in the HCN'content. -25- The Influence of Season on the HCN Content o e over er marinental Procedure ' The area selected for this experiment is located on the Central Experimental Farm, Ottawa. The soil is a good clay loam, well drained and reason bly uniform. Material used:- Plants grown in 3-inch clay pots frm selected Nappan wild white clover seed and which were trans- planted to the field in early June were used in this experi- ment. Method of Testiggv The method used in this experiment was the same as was delineated in experiment 1 except that the samples for HCN tests were taken every 10 days on 96 plants over a period of 90 days, viz., July 10th to October 8th. Climate:- The data (contained in Text Table II were furnished by the Dominion of Canada Meteorological Service from the station at Ottawa. In addition to reporting the weather conditions for the period that the plants were tested, figures are also included for the month of June, as these had a direct bearing on soil conditions at the time of the experiment . Text Table II Meteorological Data 1939 Mean Average Average Total Total hours Temperature Maximum Minimum Precipitation of bright Te . Tem . sunsh ne of a; of Inches Hours June 63.5 74.5 52.6 3.61 241.6 July 67.9 79.3 56.6 6.32 294.3 August 68.5 79.8 57.3 3.24 303.5 September 56 . 1 66 . 3 45 . 9 2 . 89 176 . 4 .October 44.0 53.1 34.9 3.02 112.8 “afiflJin .vY,lr.I4',pu 4 kru.xr..u/.uml.fi)k‘ w .- ,. -26- Results:- The following table indicates the variation in the HCN content due to climatic conditions of plants tested in the field. Text Table x: Average HCN Content of 42 Plants Tested at 106Day Intervals, over a Period of 90 Days - July 10 to October 8, 1939. (For complete data see Appendix Table V.) Dates tested Mg. HCN/c.c. July 10 .024 July 20 .024 July 30 .028 August 9 .037 August 19 .036 August 29 .034 September 8 .031 September 18 .029 September 28 .028 October 8 .027 "Nbcessary diffiiififii'for significance (P = .05) .005 N33. Actually 96 plants were tested of which 54 gave negative HCN readings through- out the test. Using the calculated necessary difference for significance (P'==.05), the EC! readings taken.on August 9, August 19, August 29, and September 8, were sig- nificant to all other dates tested. -37- The foregoing is also expressed graphically (see Fig. 2). The graph shows that there was a slight increase in the HCN content throughout the month of July up until August 9 when a seasonal maximum was reached, and that from then on the HCN content gradually decreased until the end of the experiment (October 8). Discussion:- Numerous workers have discovered a pronounced seasonal variation.1n.HCN content of different crops. In the case of wild white clover, Askew (2) merely stated that there was a marked seasonal variation. Rogers and Prykohm (31), while working with the same crop, showed an increase in the cyanogenetic power of plants with an increase in the size of plants and with progress of season. The same workers observed that the percentage of plants reacting negatively decreased from 71.28 per cent to 57.60 per cent with the advance of season, and they did not mention any particular time during the season when a maximum HCN level was reached. .As mentioned above the highest HCN content was registered on August 9. This was likely due to an abnormally heavy precipitation of 5.60 inches during the last four days of July when the average HCN level for the 42 plants tested was .02; mg. HCN per c.c. because approximately 10 days later the lave: forethe same 42 plants was found to be .037 mg. HCN per c.c. During no other period under test was a similarly rapid rise detected. If the heavy precipitation .influenced the rapid rise in HCN content, there is agreement 'with the results of experiment 1, when under greenhouse .mcnoeH ma soapspaqaoonm hawme can swap meeuom s nmbo muesam mm we pempeoo 20m omeacb< .m .mHm 0H\m m\wm m\ma m\m m\mm w\ma w\m ~\om ~\om ~\oa oo.o / /\ /\ «I owe. om.o ‘< \x- mmo. , \\ OOQH / omo. / _// // II om.a A. .1: mmo. A oo.w aoeeepaeaeeam pfmaeoo 20m .1 Z om.m _ .eexzom. seepeeaeaeeam -23- conditions adequate soil moisture caused an increase in.HCN“ content. . It is interesting to observe that the results obtained in this experiment do not support those of Rogers and Frykolm.(3l) in that, in spite of seasonal development and increase in plant size, the HON content of 'September 28 and October 8 was not significantly higher than that of July 10, the time of the first test. -29- The Influence of Temperature on the HCN Content of White Clover merment Experimental Procedure This experiment was conducted in the greenhouse with a view to ascertaining the influence of temperature on the HCN content of wnite clover. Material used:- Three plants of varying HCN content were chosen for this experiment. Each of the three plants was then divided vegetatively into nine separate units, and the pro- cedure outlined in Experiment 1 was used. These plants were started in 3-inch clay pots containing good clay loam which had been thoroughly mixed to ensure uniformity. _Treatments:- The population of 27 plants was divid- ed into three representative groups and each group subject- ed to a different temperature. Temperatures were recorded twice daily at 7.00 a.m. and 5 p.m. for both soil and atmos- phere. Text table II summarizes the temperature data for a 53 day period. Text Table II: Summary of Temperature Data during 55 day Test. Traatment Greenhouse Temperature Soil Temperature (Average for 55 days). (Average for 55 days) 7 a.m. 5 p.m. Mean 7 a.m. 5 p.m. Mean 1 65° 1 68° F 66.5° 1' 59° T 62° F 60.5° 1' 58° 1' 60° 1' 59.0° F 55° F 57° 1' 56.0° I 3 55° 1' 57° 1' 56.0° F 55° 1' 55° 1' 54.0° I Methoa of Testing” The method for testing in this experiment -30- was similar to that used in Experiment 1. Results:- The following table indicates the influence of both greenhouse and soil temperatures on the HCN content of the plants tested. The data analyzed statis- tically are presented below. Text Table III Analysis of variance for Temperature Treatments of plants at the End of 55 Days. (For complete date see Appendix Table VI.) r Value for me to Dara M08. P val“. fi.°. j: Poo. Treatments 2 .00000048 Plants 2 .00136403 284.17 4.49 8.53 Replicates 2 .00001158 3.55 4.49 8.53 Plants 1 4 .00000104 Treatments Error 16 .00000326 Treatment . Means 1V8. Temperature Level Mg. acn/ c.c. 1 ‘ Greenhouse 88.50 1: Soil 60.5° r .0232 2 ' 59.00‘3: " 56.00 r .0237 3 ' 56.0° I: ' 54.0° I .0233 necessary difference for significance (P 5 .05 .0018 (P‘- .01 1 .0025 lFer'(P = .05) there are no significant differences between «fl. .3 1.. u. . . r hr. 52“. '. ‘ 1 .I1 -31- treatments. Ior (P'= .01) there are no significant differences between treatments. It is evident that there are no significant differences due to the temperature ranges to which these plant units were submitted. Discussion:- The mean daily temperature range in the different sections of the greenhouse was from.56° r to 66.5° I and for the respective soils the range was from 54° T to 60.5° r. W At Ottawa, plants grown under . ordinary field conditions are sometimesexposed to a wide range of temperature during a 24 hour period, even during the summer season. Temperature changes of as much as 40° I to 50° r are relatively common during a full day. With this in mind, a small experiment was conducted in which a typical plant was tested for HCN content dairy over a 20 day period. During this time the average daily temperature varied frmm 510‘! to 72° I but the variation in HCN'was very slight, ranging from..026 to .030 mg. HCN per c.c. of solution. The total precipitation during the period was only 0.15 inches. From.the foregoing it appears that normal seasonal fluctuations in temperature have little or no effect on the concentration of HCN’in.whitc clover plants. It may be that very great extremes would have some influence since they might affect the growth processes of the plants. -32- In the review of literature only one reference was found on the influence of temperature on the HON content of plants. This reference by Franske et a1 (11) related to a greenhouse experiment on sorghum in which was studied the influence of a combination of two factors on the HCN content, viz., light and temperature. It was found that reduced light combined with reduced tem- perature resulted in slightly lower HCN content. -33- The Influence of Light on the HCN Content 3T_Wfiite Clover (Experiment 41 Experimental Procedure This small experiment was set up in the greenhouse during the winter of 1939-40 in order to study the influence of direct light as opposed to diffused light on the HCN'content of wild white clover. material used:- Twelve plants with varying HCN'content were selected for this test. Each plant was divided vegetative- ly into four separate units and planted in 3-inch clay pots containing good loam.soil which was thoroughly mixed to ensure uniformity. The purpose of cloning in quadruplicate was to provide replicates for two treatments. Treatments:- The plants were divided into two similar groups. One group was exposed to normal sunlight conditions in the greenhouse, while the other was enclosed in a white cotton cage which was arranged so as to permit adequate air circulation but at the same time diffuse the light which the plants received. Plants were grown under these conditions for 55 days. ' method of Testing:- The procedure for testing in this ex- periment was the same as for experiment 1. Duplicate samples were taken from.aach plant at three different dates during the course of the experiment and tested for HCN content. Results:- The following table indicates the variation in the HON content due to the influence of light. The data were treated statistically. -34- Text Table XIII Analysis of variance for light Treatments on the HON Content of plants Tested on Three Different Dates over a Period of 55 Days. (For complete date see Appendix Table VII.) Due to 13.3. ’ 11.3. 1' Value 5:33:31“; if: Plants 11 .00121896 375.06 1.93 2.51 Treatments 1 .00007803 24.01 3.98 3.98 Dates 2 .00004655 14.32 3.13 3.18 Replicates 1 .00001225 3.77 3.98 3.98 Plants x Treatments 11 .00000560 1.72 1.93 1.93 Plants x Dates 22 .00000296 Treatments x Dates 2 .00003213 9.89 3.13 3.13 Plagtgaiegreatmen“ 22 .00000588 1.81 1.69 2.11 No. Treatment mg.¥§gfi7c.c. 1 Ordinary sunlight .0279 2 Reduced sunlight .0294 Necessary difference for significance (P = .05) .00058 (P = .01) .0007? For (P ' .05) treatment 2 is significant to treamment 1. For CP . .01) ” 2 " highly significant to treatment 1. -35- No. Dates means mg. HCN/c.c. lst November 13, 1939 .0280 2nd December 11, 1939 .0282 3rd January 6, 1940 .0298 Necessary difference for significance (P = .05) .00072 (P'= .01) .00094 For (P = .05) 3rd date is significant to let and 2nd dates. For (P :..01) " ” ” highly significant to let and 2nd dates. It will be observed that significant differences were obtained in this test i.e., after a 55 day period the amount of HCN'in plants grown in controlled light was .0015 mg. per c.c. higher than that of plants grown in ordinary light. From.Appendix Table VII it may be seen that the significance between dates can be attributed chiefly to the influence of treatment 2, as there were only slight differences between dates under treatment 1. Discussion:- The data presented above reveal that reduced light produced an increase in.the HCN content, which is contrary to the findings of Franske et a1 (11) who found that reduced light combined with reduced temperature result- ed in slightly lower'HCN'content. It must be noted, however, that the above experiment was conducted over a 55 day period and that, during the first 29 days of the test, no significant -35- differences were found but that during the latter part of the period, when the days were becoming a little longer, . significant readings were obtained. Consideration.must also be given to the fact that the temperature under diffused light was often 100 F higher than in ordinary light. -37- Correlation of HCN Content with Habit of Growth In White Clover (Experimentfg) Experimental Procedure The area used for this experiment was adjacent to the area used for experiment 2. material used:- In the summer of 1938 a breeding block of 4248 plants was established. These plants were started in 3-inch clay pots in the greenhouse in early Spring, and later transplanted in the field. Method of Stu:z:- By making HCN readings on a large random.sample of the nursery plants and by utilizing the notes relating to growth type, leafiness, and leafhopper in- jury, which were made by the white clover plant breeder, it was possible to get some measure of how growth factors might govern the presence or absence of HCN. method of Testing:- The methods of sampling and testing for HCN content were the same as outlined in experiment 1, with the exception that three compound leaves instead of five were taken in sampling. The plant characters were scored in the following manner: Growth type 1 very close growing - wild type 2 semi-upright - intermediate type 3 upright - approaching common type 4 approaching mammoth type Leafiness -- scored 1 to 5 Leafhopper injury -— scored 0 to 5. -38- Results:- Three correlations were run, namely, growth type and.HCN content, leafiness and HCN content, and leafhopper inJury and HCN content. Text table XIV'summarizes the results. Text Table XIV Correlations of Plant Characters and the HON Content. Level of Significance Character n for P = 05 Fishers VtA. Table Growth type and.HCN content - .0055 .1946 Leafiness ” " " +—.Ol46 .1946 Leafhopper injury and.HCN _ content .0135 .1946 Discussion:- It is evident that the correlation co- efficients obtained are so small that they are below the level of significance. It is rather surprising that a higher cor- relation figure was not obtained between growth type and HCN’ content since other workers, such as Doak (6), have found that persistency and high production, which are governed to some extent by growth type, were correlated with high.HCN content. -39- GENERAL‘DISCUSSION In a study of this kind in which many con- tributing factors are not under control, wide variations and discrepancies in the data must be expected and allowed for in drawing conclusions. However, it has been possible in this study, by the use of a satisfactory number of replicates and by statistical analysis, to show that some factors had a marked influence on the HCN content of white clover while others had indeterminate effects. The data presented indicate that under greenhouse conditions adequate soil moisture produces a high concentration of HCN, and that restricted soil moisture re- sults_in a relatively lower concentration. This problem.was also studied under field conditions by obversation of the influence of precipitation on the HCN'content, and it was noted that heavy precipitation was followed by an abrupt rise in the HCN content. It is realized that precipitation measurements do not give so accurate an estimation of available soil moisture as soil moisture determinations taken under greenhouse conditions. The findings of some other workers have been in agreement, while others have drawn opposite con- clusions, but it must be noted that crops such as Sudan grass and sorghum, and not white clover, were studied and that, therefore, water requirements and habit of growth were much different. Experiments have been reported where a pronounced seasonal variation in.HCN content of wild white -40.. clover was found. This is in general agreement with the rer sults of this study which, in addition, have revealed that there is a period during the growing season when a maximum amount of HCN is present. A study of temperature effects showed a relatively unaltered.HCN content over a range of temperature. It is readily seen that there were so many uncentrollable factors present in this part of the study that the findings must be considered inconclusive. It is evident that moderate changes in temperature, especially if the changes were constant and plants exposed to them for fairly long periods, would definitely affect the growth processes of the plants and pro- vide a condition in which changes in.HCN content might be expected. In considering the influence of light on the HCN content, the difficulty of securing satisfactory con- trol must be emphasized. It was found that diffused light augmented the HCN content significantly. This finding is qualified, however, since a 55 day period, during which the daily duration of light increased, was necessary to obtain signiffcant results. Franske et a1 (11), as stated previously, found that reduced light combined with reduced temperature caused a slight reduction.in.HCN content in Sudan grass. Perhaps of most interest is the failure to obtain significant correlations between different plant characters and HCN'content. Other investigators have de- lnonstrated that in white clover the HCN'content is directly -41— correlated with persistence and productivity, but in this study where a fairly large sample was taken no correlation was established. 0n the whole, this study must be con- sidered to be of an exploratory nature, inasmuch as many unpredictable factors showed up to condition the results. The findings may nevertheless serve as a useful guide to more detailed work. 3. 5. -42- summer Adequate soil moisture produced a high concentration of HCN in wild white clover, and restricted soil moisture resulted in a relatively lower concentration. There were a pronounced seasonal variation in the HCN content of wild white clover and also an indication of a period of maximum HCN'content. The influence of temperature on the HCN content was found to be statistically insignificant. It was found that diffused light augmented the HON content significantly in comparison to ordinary light. There were no significant correlations found between the plant characters studied and the HCN'content. ACKNOWLEDGMENTS The writer wishes to acknowledge his in- debtedness to Dr. T. M; Stevenson for putting the facilities of the Division of Forage Plants at his disposal; to Dr. J. W. Hopkins for statistical help; to Mr. R. M. MacVicar, Hr. F. S. Nowosad, and Dr. J. M; Armstrong for their assistance and constructive criticism in the preparation of this paper. 1. 2. 3. 4. 6. 7. 8. 9. 10. 11. 12. 13. 14. -43- REFERENCES Armstrong, H. E., E.F. Armstrong, and E. Horton. Herbage Studies II - variation in Lotus corniculatus and Trifolium repens (Cyanophoric Plants). Proc. of the Royal Society, London B. 86 : 262-269. 1913. Askew, H. 0. Determination of hydrocyanic acid in white clover. N. Z. Jour of Sci. and Tech. 15 : 227-233. 1933. Boyd, F. F., 0. S. Aamodt, T. Bohstedt, and E. Truog. Sudan grass management for control of cyanide poisoning. Brflnnick, J. C. Hydrocyanic acid in fodder plants. Jour. of Chem. Soc. Transactions 28 : 788-796. 1903. Coleman, 0. H., and D. W.‘Robertson. Colo. Agr. Exp. Sta. Tech. Bull. 24. 1938. Doak, B.‘W. .A chemical method for the determination of type in white clover. N. Z. Jour of Sci. and Tech. 14 : 359-365. 1933. Dowell, C. F. Cyanogenesis in Androgon sorghum. Jour. Dunstan,‘w. R., and T. A. Henry. The chemical aspect of cyanogenesis in plants. Brit. Ass. Ad. 30., p. 145. 1906. Foy, N. 3., and E. 0. C. Hyde. Investigation of the re- liability of the "picric-acid test” for distinguish- ing strains of white clover in New Zealand. N. Z. Jour. of Agr. 55 : 219-224. 1937. Francis, C. K. .Poisoning of livestock while feeding on plants of the sorghum group. Okla. Agr. Exp. Sta. Ciro. 38. 1915. Franzke, C. J., L. F. Puhr, and A. N. Hume. .A study of sorghum.with reference to the content of HCN. S. Dakota State College Tech. Bull. 1. 1939. Greshoff, M. The distribution of prussic acid in the vegetable kingdom. Brit. Ass. Ad. Sci., p. 138. 1906. Hindmarsh, W. L. Some Australian poisonous plants : amounts fatal to sheep. Jour. of Council of Sci. Ind.‘Res., p. 12. 1930. Leeman, A. C. Hydrocyanic acid in grasses. 0nd. Jour. Of Vet. 3021. and An. Inde 5 : 97-136e 1935e 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 29. -44- Manges, J. D. Vet. med. 3O : 347-349. 1936. Maxwell,‘w. Sorghum.poisoning. Queensland Agr. Jour. 15 : 473. 1903. Marias, J. S. 0., and C. Rimingtcn. Isolation of the poisonous principle of Dimorphoteca cuneata, Less. 0nd. Jour. 3 : 111. 1934. Menaul, P., and C. F. Dowell. Cyanogenesis in Sudan grass : a modification of the Francis Connel method of determining hydrocyanic acid. Jour. Agr. Res. 18 : 447-450. 1920. Miranda, M. M. Sur la presence de l'acide cyanhydrique dans le trefle rampant. Compte rendu de l'acad. de Sc. Paris 155 : 651. 1912. Narasimha Acharya, C. Investigations on the development of prussic acid in cholam (Sorghum vulgare). Indian Nowosad, F. W., and R. M. MacVicar. Adaptation of the ”pioric-acid test" method for selecting HCN-free lines in Sudan grass. Sci. Agr. 20 : 566-569. 1940. Pease, H. T. Poisoning of cattle by.Andropogon Sorghum. Jour. Comp. Med. and Vet. Arch. 18 : 679. 1897. Petrie, I. M. Hydrocyanic acid in plants. Part 2. Its occurrence in the grasses of N. S. wales. Linn. Soc. N. 8. Wales, p. 624. 1913. Peters, A. T., H. B. Slade, and S. Avery. Poisoning of. cattle by common sorghum and kaffir corn. Nebr. Agr. Exp. Sta. Bull. 77. 1903. Pethybridge, Geo. H. Is it possible to distinguish the seeds of wild wnite clover from.those of ordinary white clover by chemdcal means during a germination test? Roy. Dublin.Soc. 2 : 248-258. 1919. Progress report of pasture investigation. Canada, Dept..Agr., Ottawa. 1939. Ramsay, A. A., and M. Henry. Rosewood (Heterodendron oleaefolium) and native fuchsia (Eremphila maculata) two poisonous plants. Agr. Gazette N. S.‘Wa1es 4O : 834. 1929. Ravenna, C. E. Peli. Gaz. Chim. Ital. 37 : 568. 1907. ‘Rigg, T., H. 0. Askew,and‘E. B. Kidson. Occurrence of cyanogenetic glucosides in Nelson pasture plants. N. Z. Jour. of Sci. and Tech. 15 : 222. 1933. -45- Sudan grass and other Rogers, C. P.,and W. L. Boyd. Jour. of 30. cyanOphoric plants as animal intoxicants. 489-500. 1936. Amer. Vet. Med. Assoc. 88 . 31. Rogers, Chas., and O. C. Frykolm. Observation on the variations of cyanogenetic power of wnite clover 533-537. 1937. plants. Jour. of Agr. Res. 55 : 32. Sampson, K. Cyanophoric tests with seedlings and plants of white clover. Welsh Plant Breeding Sta. Bull. 33. Seddon, H. R.,and R. O. C. King. The fatal dose for sheep of cyanogenetic plants containing sambunigrin 3 : 14. or prunisah. Jour. Council Sci. Ind. Res. 1930. 34. Sullivan, J. T. 3rd Ann. Rpt. U. 3. Regional Pasture State College, Pennsylvania. 1939. Res. Lab. Hydrocyanic acid in Soudan grass. 35. Swanson, C. O. Agr. Res. 22 : 125. 1921. 36. Vinall, H. N. A.study of the literature concerning poisoning of cattle by prussic acid in sorghum, Sudan grass and Johnson grass. Jour. Amer. Soc. Agron. 13 : 267. 1921. 37. were, W. M. Experiments and observations on forms and strains of Trifolium repens L. Jour. of Agr. Sci. 15 ' 47-67. 1925. O Jour. Notes on the hydro- 38. Willaman, J. J.,and R. M. West. cyanic content of sorghum. Jour. Agr. Res. 4 . 179-185. 1915. 39. ‘Willaman, J. J.,and R. M. West. Effect of climatic factors on the hydrocyanic-acid content of sorghums (Sorghum.vu1gare). Jour. Agr. Res. 6 : 261-365. 1939. 40. ‘Williams,‘R.‘D. Genetics of cyanogenesis in white clover (Trifolium repens). Jour. of Genetics 38 : 357-365. 1939. Amp. Table 1. Summary of data from Experiment 1. Kg. HCN per cc. for group "high" in HCN content. Treatment 1 Treatment 2 ireatment 3 Plant Rep.1 Rep.2 nep.3 Rep.1 Rep.2 Rep.3 Rep.1 nep.2 Rep.3 1 .060 .053 .059 .042 .041 .037 .032 .015 .018 2 .042 .041 .040 .040 .041 .040 .027 .028 .027 3 .043 .044 .045 .039 .039 .034 .031 .027 .029 4 .037 .038 .046 .035 .036 .037 .030 .026 .027 5 .041 .043 .042 .040 .038 .040 .031 .029 .028 App. Table 2.- Summary of data from EXperiment 1. Mg. HCN per cc. for group "medium" HCN content. Treatment 1 Treatment 2 Treatment 3 Plant Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 1 .035 t034 .036 .031 .030 .032 .028 .027 .028 2 .044 .042 .044 .041 .042 .040 .038 .036 ..037 3 .027 .025 .025 .024 .023 .023 .020 .021 .018 4 .026 .023 .025 .024 .024 .023 .022 .020 .019 5 .022 .019 .021 .019 .020 .019 .016 .015 .017 App. Table 3. Summary of data from.Experimcnt 1. Mg. HCN per cc. for group "16w " HCN content. Treatment 1 Treatment 2 Treatment 3 Plant Rep.1 Hep.2 Rep.3 Rep.1 Rep.2 Rep.3 Rep.1 Rep.2 Rep.3 1 .016, .012 .011 .014 .013 .014 .015 .014 .0112" 2 .012 .010 .011 .010 .009 .009 .009 .000 .000 3 .022 .022‘ .023 .021 .021 .022 .020 .022 .022 4 .026 .025 .025 .024 .026 .026 .022 .024 .026 5 .009 .009 .012 .010 .009 .009 .000 .009 .000 2.3.8 o a... . ......,.. .mm........ a. a... a... 3.. -3. 3.. 3.--..3... .3? 83%.. .8. o... .2. .8. .8. .8. .8. .8. a... .8. .8. o... .8. .1»... .8. .8. e8. .8. ooo. .8. ooo. .8. .8. .8. a... .8. .8. o 8.. a... .8. 8o. .8. 8o. «8. .8. 8o. .8. .3. Bo. .8. o .88 o... 8. 8o. .8. .8. 3o. o8. a... .8. .8. 8o. .8. «no. a 8.. o... .8. 8o. .8. ooo. ooo. .8. .8. .oo. o... ..o. o... a... a .8 3.. .8. .8. .8. .8. . . . 2... ‘78.. 1 a... .3. a... .... 8.. .3. o... .2. .2. 3m. 8.. 3.8. 8.. . 8o. .8. .8. a... .8. .8. 8o. .8. .8. .8. .8. a 8.. .8. .8. 8o. .8. o... .8. .8. 8o. 8.. .8. .8. 8o. . .8. .8. o... ..o. e... .8. .8. lo. .8. 8o. .8. 8o. .8. a 8.. 8.. .... a... .8.. .8. .8. 8o. .8. .8. .8. 8o. .8. .8. a 5...: 0 0 30. d .8.. 8.. 2... 2... 8.. 8.. .2. 8.. o... 8.. .2. .8. a... 38.. .8. .8. e... 8.. .8. .8. .8. 2... 8o. .8. .8. n8. .8. o a... .8. 8.. .3. .8. 8o. .8. .8. .8. 8o. .8. 8o. .8. o 88. .8. .8. a... a... .8. .8.. .8. 8o. .8. .8. .8. .8. .8. n no: In. .8. do... 8.. .8. 8o. .8. .8. .8. 8. 08. .8. .8. u no... 3.. .8. o... a... 3on1 a... 8. .8. .8. a.m. .8. n8. .8. . n a . 2.38.81.8- ...8 «.2... .8.- ...s. a... .8.- 3...... 3.8.. 3.33.1... .82... 1 : gala-.5 n awe-bee.“ u val-sub A uncles-h B «.3... Too no... 2%. .ma .mmoonw .30.?- ooe .8530...- .-..m.E- made... you 9.3. 01. no one no 2.3.8.. 20.. on... so 0.33.08 .30. no 02.2.3... 05. .H 5083038 80.... some no has-58m 3.. 0.389 .9... App. Table 5. Summary of date from experiment 2. The nCN content of 96 plants tested at lO-dey intervals over a period of 90 days.(Mg. HCN per cc.) Clonal July J uly July Aug. Aug . Lug . Sept . Sept . sept . act . Row No. 10 20 29 9 19 29 8 18 28 8 1 .035 .034 .033 .045 .044 .044 .043 .043 .042 .040 2 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 3 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 4 .000 -000 .000 .000 .000 .000 .000 .000 .000 .000 5 .022 .020 .024 .033 .030 .025 .023 .024 .024 _.024 3 .024 .025 .023 .033 .032 .030 .050 .025 .023 .027 7 .045 .047 .050 .057 * .054 .052 .052 .050 .050 5 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 9 .012 .015 .015 .030 .030 .024 .022 .020 .015 .015 10 .050 .051 .052 .059 .055 .054 .054 .052 .052 .054 11 .024 .025 .025 .035 .032 .032 .025 .025 .027 .025 12 .030 * .034 .042 .042 .040 .035 .034 .034 .030 13 .009 .005 .009 .013 .014 .010 .010 .010 .010 .010 14 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 15 .035 .040 .045 .045 .047 .044 .042 .042 .040 .040 13 .039 .042 .042 .043 .044 .044 .042 .040 .042 .040 17 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 15 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 19 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 20 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 21 .015 .015 .020 .024 .022 .020 .017 .017 .017 .017 22 . * .023 .025 .034 .032 .030 .025 _.025 .023 .023 23 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 24 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 25 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 23 .034 .033 .035 .040 .035 .033 .034 .033 .034 .034 27 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 25 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 29 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 30 .014 .013 .013 .033 .030 .023 .031 .024 .020 .015 31' .017 .017 .024 .025 .030 .023 .024 .020 .020 .015 32 .034 .032 .034 .044 .044 .040 .035 .033 .033 .032 33 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 34 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 35 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 33 .014 .015 .013 .022 .020 .020 .014 .013 .012 .012 37 .013 .017 .023 .025 .034 .032 .025 .019 .015 .005 35 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 39 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 40 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 41 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 42 .022 .020 .024 3045 .039 .035 .037 .032 .030 .030 43 .032 .033 .041 .043 .041 .043 * .035 .033 .032 44 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 45 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 43 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 47 .020 .021 .025 .045 .043 .040 .025 .027 .027 .023 45 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 (cont'd) 49 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 50 .012 .013 .022 .032 .025 .023 .022 .020 .015 .014 51 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 52 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 53 .013 .013 .024 .033 .032 .025 .023 .024 .022 .020 54 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 55 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 53 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 57 .017 .015 .024 .032 .030 .025 .023 .022 .020 .022 55 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 59 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 30 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 31 .005 .009 .012 .022 .022 .015 .013 .010 .010 .010 32 .042 .045 .044 .055 .053 .054 .054 .052 .050 .050 33 .014 .014 _.015 .027 .023 .024 .024 .015 .020 .020 34 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 35. .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 33. .012 .013 .027 .027 .027 .024 .022 .022 .020 .013 37 .040 * .043 .055 .053 .055 .050 .052 .043 .044 35 .017 .015 .020 .025 .033 .025 .025 .024 .022 .020 39 .013 .014 .020 .032 .033 .030 .033 .023 .023 .022 70 .009 * .005 .019 .014 .014 .012 .010 .009 .009 71 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 72 .010 .012 .011 .022 .020 .015 .014 .013 .013 .014 73 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 74 .025 .023 .037 .044 .042 .040 .035 .035 .035 .034 75 .033 .034 .033 .052 .050 .045 .045 .044 .042 .042 73 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 77 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 75 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 79 .015 .015 .017 .033 .032 .023 .020 .020 .015 .015 50 .040 .037 .042 .034 .054 .044 .043 .042 .040 .033 51 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 52 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 53 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 54 .015 .013 .022 .032 .035 .034 .033 .022 .020 .020 55 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 53 .034 .033 .042 .052 .045 .044 .040 .033 .033 .033 57 .034 .033 .034 .042 .044 .042 .040 .040 .033 .035 55 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 59 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 90 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 91 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 92 .024 .023 .032 .039 .035 .034 .032 .030 .025 .030 93 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 94 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 95 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 93 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 Mean .024 00“ 0028 0037 .036 0034 .031 .029 .038 0087 . 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