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T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
This identification was confirmed by Dr. van Suchtelen of t h e Department of Microbiology. The source of t h e infection by the microorganism is not known b u t t h e facts outlined in t h e first paragraph t h a t only one barrel out of a possible hundred develops t h e organism a n d t h a t t h e barrels used are necessarily second-hand, point t o the previously inoculated barrel as t h e source of t h e trouble. As it appears unusual t h a t these microorganisms should appear in quantities in lime-sulfur concentrates d a t a regarding the mixture may be of interest. Density.-The densities of t h e ketchup mixture, t h e filtrate from it a n d of other lime-sulfur solutions were taken in a n ordinary specific gravity bottle of 2 j cc. capacity, with a n open capillary tube, a t room temperature which was z j ' . The results are recorded in t h e following table: TABLEI1
No. 1
2 3 4
MATERIAL Water Lime-sulfur ketchup Lime-sulfur solution Lime-sulfur solution
6
Lime-sulfur solution Lime-sulfur solution
7
Lime-sulfur solution
8
Lime-sulfur solution
5
B. from Observed commercial density scale REMARKS 0.9966 . . . . Check 1.3047 34.0 1.2996 3 3 . 6 Filtrate from Expt. 2 1.3020 33.7 Ketchup heated until solution resulted and loss of volume made up 1.2992 3 3 . 6 Duplicate of No. 4 1.3214 3 5 . 3 Ketchup heated until solution resulted and loss of volume not made up 1.3Oi8 34.2 Same as iVo. 6 except heated with air condenser 1.3072 34.2 Same as h-0. 6 except heated under return water condenser
An examination of t h e data in Experiments 7 and 8 shows t h a t t h e original lime-sulfur solution in which t h e microorganisms developed was of t h e maximum density, 1.3075 (34' B.), for commercial preparations, and Experiment 3, t h a t the filtrate from t h e ketchup was of a density of 1.2996 (33.6' B.), which is still slightly stronger t h a n necessary for t h e trade. It is further seen, Experiments 2 , 7 and 8, t h a t t h e density of t h e clear liquid is only slightly altered (-0.008) by t h e withdrawal of sulfur by t h e microorganisms. Experiment 6 as compared with 7 and 8 shows how t h e density of these concentrated solutions is changed ( + O . O I S ) by the withdrawal of a small amount of water-less than 1/2+/b~ of the total volume in this case. Suljur in Suspension -The amount of sulfur per cubic centimeter in suspension in the bodies of t h e microorganisms in t h e ketchup is 0.0043 gram as obtained from Experiment 2 in Table I. SUMMARY
Llicroorganisms of Beggiatoa, or a closely related group, have been found t o be the cause of a n apparent precipitate of sulfur occurring in commercial limesulfur concentrates, making the whole mass of t h e consistency of thin ketchup. Some d a t a regarding this thickened lime-sulfur is given. The further study of t h e microorganisms, their iden-
Vol. 5 , No.
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tification, the sources of infection, a n d means of prevention are being investigated. DEPARTMENT OF GENERALA x D AGRICULTURAL CHEMISTRY MASSACHUSETTS AGRICULTURAL COLLEGE,AMHERST
THE ASSAY OF INDIVIDUAL PLANTS OF DATURA STRAMONIUM L., DATURA TATULA L. AND OTHER SPECIES AND VARIETIES' By F. A. MILLER AND J. W. MEADER
I t has been stated in a recent communicationZ t h a t t h e examination of individual plants of the daturas for their total alkaloidal content has been undertaken as a means of following t h e effects of prolonged cultivation upon the percentage of alkaloids, a n d as a means of selecting high-yielding individual plants for breeding purposes. The present discussion is a continuation of this investigation, together with additional data upon other species a n d varieties not heretofore included. A review of t h e literature on stramonium \vas given in t h e original communication. This was followed b y t h e assays of several individual plants of Datura stramonium L. and Datura tatula L. Seeds were collected from all t h e assayed plants a n d those from the individuals containing t h e highest and lowest percentages of alkaloids from each of t h e two species were planted M a y 30, 1 9 1 2 . The results obtained from these plantings are considered in t h e present discussion. The conditions of growth were practically the same as those under which t h e parent plants were produced. The soil, a stiff clay loam though not identical, was t h a t of a n adjacent field a n d of a similar physical character. The plot was fairly uniform except for an excess of sand and gravel a t one end. The plants growing a t this point were smaller a n d less vigorous t h a n t h e others. When about three inches high they were thinned t o four feet in t h e row. Cultivation was begun a t this time a n d continued throughout t h e gromring season. Eight plantings were made, four of Datura stramonium L. and four of Datura tatula L. These were made from t h e highest a n d lowest yielding individuals from each of t h e two species, as given in t h e original communication. The plants indicating these extremes assayed 0 . jj per cent and 0.46 per cent in Datura stramonium L. a n d 0 . 6 5 per cent and 0.47 per cent in Datura tatula L. Four of t h e above plots received a n application of normal commercial fertilizer a t t h e rate of six hundred pounds per acre. This was applied when t h e plants were about one foot high. The collection of samples of leaves for assay from individual plants of each of t h e eight plots was made early in September. These samples were dried a t room temperature, granulated and sealed in amber bottles until assayed. Following are t h e results obtained from nineteen selections of individual plants from Datura stramonium L. Ten of these were from t h e high- and nine from the low-yielding parent. 1 Paper presented a t the 47th meeting of the A. C. S.. Milwaukee, March, 1913. 2 8th In1 Cong. A s p . Chemislry, 17, 57.
I Dec., I913
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y T A B L EI-Datura
stramonium L.
Highest plant originally assayed 0.55 per cent
-
Without fertilizer
Source
h'o.
B- 980 B-1349 B-1351 B-1352 B-1353 B-1354
With fertilizer 7
Parent plant Selection Selection Selection Selection Selection
Assay Per cent
No.
0.55 0.50 0.56 0.50 0.54 0.42
B- 980 B-I360 B-1361 B-1362 B-1363 B-1364
__
Source Parent plant Selection Selection Selection Selection Selection
Average, 0 . 5 0
Assay Per cent 0.55 0.67 0.66 0.62 0.65 0.48
__
Average, 0 . 6 1
Lowest plant originally assayed 0.46 per cent Without fertilizer
With fertilizer c
3-0.
B- 982 B-1355 B-1356 B-1365 B-1366 B-1367
Source Parent plant selection Selection Selection Selection Selection
Assay Per cent 0 46 0.58 0.57 0 58 0.65 0.63
so.
B- 982 B-1357 B-1368 B-1369 B-1370
Source Parent plant Selection Selection Selection Selection
__
Assay Per cent 0.46 0.64 0.68
0.58 0.71
-
Average, U . 64
Average, 0 . 6 0
T h e Datura fatula L. selections, of which there were t e n each from t h e high- a n d low-yielding parents, gave t h e following results: T A B L E11-Datura
tatula L.
Highest plant originally assayed 0.65 per cent XVithout fertilizer With fertilizer
No. B- 984 B-1344 B-1348 B-1376 B-1377 B-1383
Source Parent plant Selection Selection Selection Selection Selection
Assay Per cent 0.65 0.53 0.56 0.77 0.59 0.64
-
No. B- 984 B-1378 B-1379 B-1380 B-1381 B-1382
Source Parent plant Selection Selection Selection Selection Selection
Average 0 . 6 2
Assay Per cent 0 65 0.72 0.75 0.69 0.73 0.44
Average, 0 . 6 8
Lowest plant originally assayed 0.47 per cent LVithout fertilizer ll'ith fertilizer r N O .
B- 985 B-1341 B-1342 B-1343 B-1346 B-1347
7
Assay Per cent
Source Parent plant Selection Selection Selection Selection Selection
0.47 0.47 0.45 0.52 0.45 0.5i
NO.
B- 985 B-1371 B-1372 B-1373 B-1374 B-1375
Source Parent plant Selection Selection Selection Selection Selection
Assay Per cent 0.47 0.66 0.50 0.48 0.53 0.54
Average, 0 . 5 4
Average, 0.49
A small plot of Datura ferox L. was grown from seed obtained from Haag & Schmidt, of Erfort, Germany. F o u r selections were m a d e a n d samples of leaves from these selected plants assayed as follows: T A B L EIJI h-0.
B- 1345 B--1350 B-1358 B-1359
Source
Datura ferox Datuva ferox Datura ferox Datura ferox
Assay Per cent 0.53 0.62 0 TO 0.66
A small planting of Datura humulus j7at,a was made of seed from t h e above source. A mixed sample of leaves assayed 0.4 2 per cent. Individual selections
IO1 j
could n o t be made o n account of t h e failure of t h e plants t o produce seed. This was due t o t h e late planting a n d injury from early frost. T h e plants did not flower until September a n d were extremely sensitive t o frost. T h e foregoing figures indicate a considerable range of variations in t h e percentage of alkaloids as found in t h e individual plants. This variation i n t h e progeny of both t h e Datura stramonium L. a n d Datura tatula L. selections is greater t h a n t h a t of t h e t w o parent plants. I n t h e species stramonium this is 0.09 per cent in t h e parents, while i n t h e progeny i t is 0.23 per cent for t h e selections from both t h e fertilized a n d non-fertilized plots. T h e highest yielding plant was produced b y t h e low parent upon a fertilized plot while t h e lowest yielding plant was produced b y t h e high parent upon a non-fertilized plot. Contrary t o expectations t h e higher averages were obtained upon t h e fertilized a n d non-fertilized plots from t h e low-yielding parent, F o r t h e fertilized plots these averages are 0.64 per cent f r o m t h e low parent a n d 0.61 per cent from t h e high parent. For t h e non-fertilized plots t h e y are 0 . 6 0 per cent from t h e low parent a n d 0 . j o per cent from t h e high parent. I n t h e species t a t u l a t h e variation of t h e t w o parent plants is 0.18 per cent while in t h e progeny i t is 0.31 per cent a n d 0.32 per cent upon t h e fertilized a n d nonfertilized plots, respectively. T h e highest yielding plant was produced by t h e high parent upon a nonfertilized plot, while t h e lowest yielding plant was also produced b y t h e high-yielding parent b u t upon a fertilized plot. T h e latter was a n abnormal plant producing few leaves a n d for this reason might be expected t o give a low yield. T h e next lowest appears, according t o expectation, in t h e progeny of t h e low parent a n d upon a non-fertilized plot. T h e averages i n t h i s case are t h e reverse of w h a t was noted for t h e species stramonium a n d are according t o expectations. T h e higher averages are obtained f r o m t h e progeny of t h e high parent a n d from t h e fertilized plots. For t h e fertilized plots these averages are 0.68 per cent from t h e high p a r e n t a n d 0.j4 per cent from t h e low parent. For t h e non-fertilized plots t h e y are 0.62 per cent from t h e high parent a n d 0.49 per cent from t h e low parent. Table I V shows extremes as noted in t h e parents a n d offsprings a n d t h e variations as found upon fertilized a n d non-fertilized soils. T h e greater range of variations i n individual plant yield as f o u n d i n these second generation plants is probably d u e t o t h e f a c t t h a t t h e p a r e n t plants represented mixed strains. Such mixed strains resulting from promiscuous pollination might produce plants giving altogether different yields from t h e parents. When t h e original selections were made no precautions were t a k e n t o prevent cross pollination. This precaution is necessary with t h e d a t u r a s if known parentage is desired. It has not been conclusively shown t h a t t h e property of a plant t o produce a definite percentage of alkaloids is uniformly transmitted t o t h e offsprings. Inbreeding or close pollination of all selected plants is necessary in t h e s t u d y of t h e transmission of such i I
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T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
a character. The results so far obtained indicate t h a t t h e seeds from open pollinated plants are either of composite parentage or t h a t t h e character under consideration is not uniformly inherited. The results TABLE I V Datura stramonium Assay Assay Highest yielding Lowest yielding plant plant Per cent Per cent 0.55 . 0.46 Parent plant.. Non-fertilized.. . . . . . . . . . . 0 . 6 5 0.42 0.48 Fertilized.. . . . . . . . . . . . . . . 0 . 7 1 Datura tatula 0.65 0.47 Parent plant.. 0.45 Non-fertilized . . . . . . . . . . . 0.77 0.44 Fertilized.. . . . . . . . . . . . . . . 0 . 7 5
...........
...........
Variations Per cent 0.09 0.23 0.23 0.18 0.32 0.31
seem t o point toward t h e former supposition with certain exceptions. Some degree of uniformity is t o be noted in t h e assays of these second generation plants. There is more uniformity among t h e individuals within t h e groups assayed t h a n there is between these individuals a n d t h e parent plants. Considering only t h e non-fertilized plots in this respect t h e progeny of the high-yielding stramonium is most uniform. T h e parent assayed 0 . 5 5 per cent a n d t h e progeny 0 . 5 0 per cent, 0.56 per cent, 0.54 per cent a n d 0.42 per cent. This uniformity within t h e groups is greatest in t h e t a t u l a selections and is sufficient t o maintain t h e averages in t h e order expected. Taking the average assay of t h e progeny of t h e low-yielding parent from t h e non-fertilized plot as a unit, t h e averages from t h e other plots of this species follow in their expected order, i. e., 1.00 : 1.10 : 1.26 : 1.38. I n t h e species stramonium, however, t h e uniformity within t h e groups is not sufficient t o maintain this order. I n this case t h e order is 1.00 : 1.06 : 0.82 : 1.01. Greater extremes in t h e alkaloidal yield of t h e individual plants have been located t h a n were found i n t h e original investigations. This is doubtless due t o t h e small number of plants originally assayed. It is believed t h a t such extremes can be determined immediately by t h e examination of a sufficiently large number of individual plants produced under uniform conditions. Whether or not t h e greatest extremes in t h e t w o forms under discussion have been reached can be determined only by further study. The location of t h e highest extremes is t h e principal object. T h e possibility of t h e fixation of this high-yielding property must then be determined by inbreeding. If such a procedure is possible, high-yielding strains or races of t h e alkaloidal producing plants can be developed for commercial purposes. With four exceptions all of t h e forty-three assayed plants have been inbred a n d mature seeds obtained from t h e m for planting. T w e n t y crosses (hybridizations) have also been made between various individuals of the three species studied. A few reciprocal crosses have been made. The alkaloidal yield of these hybrid forms will be studied in comparison with t h e yield of t h e inbred parents a n d their progeny. Perfect uniformity in t h e behavior of any character cannot be expected in t h e first generation from inbred plants. This is due
Vol. 5 , No.
12
t o latent tendencies, t h e elimination of which may require two or more generations b y inbreeding.
It has already been stated t h a t another object of this investigation was t o follow t h e effects of prolonged cultivation upon t h e yield of alkaloids. T h e idea t h a t cultivation causes a decrease in t h e value of medicinal plants has been current for many years, b u t apparently without foundation. The results obtained are contrary t o this belief a n d are a n indication of what may be accomplished through cultivation a n d improvement. All plants in t h e experimental plots have been under cultivation two years. T h e original source of the seed from which the cultivated strains were started is known absolutely in t h e case of Datura tatula L., as wild plants a few inches high were transplanted from a vacant lot in Indianapolis t o t h e experimental plot. T h u s two years under cultivation is known t o be correct for this species. This is not true, however, with Datura stramonium L. The seeds from which t h e original planting was made were obtained in t h e London market a n d may or may not have been gathered from wild plants. Mixed samples were collected for comparison from wild plants found growing ’ adjacent t o t h e experimental plots. Following is a table of t h e average assays of t h e wild and cultivated plants: TABLEV-AVERAGE ASSAYS (PERCENTAGES) Datura slramonium Source 1910 1912 Original plants.. . . . . . . . . . . . 0 . 5 0 Fertilized 0.61 High-yielding plant, . . . . . . . . . . . . 0.64 Low-yielding plant. . . . . . . . . . . . . Wild p l a n t s . . . . . . . . . . . . . . . . . . . . .... Datura f a l U b Fertilized Original plants.. . . . . . . . . . . .0 . 5 8 0.68 High-yielding plant.. . . . . . . . . . . . Low-yielding plant. . . . . . . . . . . . . 0.54 .... Wild plants.. . . . . . . . . . . . . . . . . . .
1912 Non-fertilized 0.50 0.60 0.28
Non-fertilized 0.62 0.49 0.35
These averages indicate t h e beneficial effect of cultivation both with a n d without fertilizer. I n most cases these averages are higher in t h e second year t h a n in t h e first. T h e general averages of all t h e selected plants assayed this year is greater in each case t h a n t h e averages of t h e plants originally assayed. The exceptions are most marked in t h e averages of t h e progeny from t h e low-yielding parent. I n Datura stramonium t h e averages are equal t o or greater t h a n t h e original average. These forms will be continued under cultivation. Seed from t h e highest yielding individuals will again be planted a n d t h e resulting plants assayed in t h e same manner as those previously examined. I n addition t o t h e original objects of t h e investigafion, i. e., t h e effects of cultivation a n d selection upon yield of alkaloids, t h e influence of hybridization upon this yield will also be noted. Other species a n d varieties will be :included as seed supplies become available. DEPARTMENTS OF BOTANYAND ANALYTICAL CHEMISTRY ELI LILLY & COMPANY, INDIANAPOLIS
