J 191 12 W5 >py 1 THE EFFECTS OF KEROSENE AND OTHER PETROLEUM OILS ON THE VIABILITY AND GROWTH OF ZEA MAIS BY JOHN HAMILTON WHITTEN A.B., University of Illinois, 1911 A.M., University of Illinois, 1912 THESIS Submitted in Partial Fulfilment of tne Requirements for the Degree of DOCTOR OF PHILOSOPHY IN BOTANY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS 19 14 - } 50 1 100 98 50 5 min. >} 50 1 100 98 Check 25 25 1 100 96 2 50 10 min. 3 da. air 50 100 100 50 15 min. )> 50 100 100 50 30 min. >) 50 3 100 94 Check 25 25 1 100 96 3 50 1 hr. 3 da. air 50 1 100 98 50 2 hr. > > 50 100 100 50 3 hr. ) > 50 1 100 98 50 6 hr. >> 50 2 100 96 50 8 hr. )} 50 100 100 50 14 hr. >> 50 100 100 Check 50 50 100 100 4 50 1 da. 3 da. air 50 100 100 50 3 da. >> 50 2 100 96 50 6 da. > 1 50 100 100 Check 25 25 100 100 4a 100 1 da. 5 da. air 100 2 100 98 100 3 da. j ? 100 1 100 99 100 6 da. j ■> 100 2 100 98 Check 25 25 100 100 5 100 10 da. 5 da. air 92 5 92 87 Check 25 25 100 100 6 100 25 da. 5 da. air 85 3 85 82 Check 25 25 100 100 7 100 30 da. 5 da. air 86 10 86 76 Check 25 24 96 96 8 100 50 da. 5 da. air 76 3 76 73 Cheek 25 25 100 100 9 100 120 da. 5 da. air 72 5 72 67 Check 25 25 100 100 10 100 158 da. 5 da. air 64 64 64 Check 25 25 1 100 96 11 100 190 da. 5 da. air 57 57 57 Check 25 25 100 100 12 100 215 da. 5 da. air 60 2 60 58 Check 25 25 100 100 13 100 1 yr. 5 da. air 66 66 66 Check 25 25 1 100 96 14 100 2 yrs. 5 da. air 56 56 56 253 Series A — Continued Table 2. Boone County White Corn in a 50% Saturated Soil Trial No. of grains Kerosene treatment After treatment No. germ. No. in- jured Per ct. germ. Per ct. norm, growth 1 Check 50 50 50 25 dipped 1 min. 5 min. 3 da. air 41 44 45 25 1 1 1 82 88 90 100 80 86 88 100 2 Check 50 50 50 25 10 min. 15 min. 30 min. 3 da. air >> 40 44 42 24 1 1 1 80 88 84 96 80 86 82 92 3 Check 50 50 50 50 50 50 1 hr. 2 hr. 3 hr. 6 hr. 8 hr. 3 da. air ;> > y j > >} 43 40 38 36 38 49 2 2 2 1 1 1 86 80 76 72 76 98 82 76 72 70 74 96 4 Check 50 50 50 25 1 da. 3 da. 6 da. 3 da. air 36 37 33 25 1 2 1 1 72 74 66 100 70 70 64 96 5 Check 100 25 10 da. 5 da. air 60 25 1 60 100 58 100 6 Check 100 25 25 da. 5 da. air 61 25 8 61 100 53 100 7 Check 100 25 30 da. 5 da. air 62 24 3 62 96 59 96 8 Check 100 25 50 da. 5 da. air 60 25 3 60 100 57 100 9 Check 100 25 120 da. 5 da. air 56 24 1 56 96 56 92 10 Check 100 25 158 da. 5 da. air 56 25 1 56 100 55 100 11 Check 100 25 190 da. 5 da. air 53 100 53 100 53 100 12 Check 100 25 215 da. 5 da. air 47 23 2 47 92 45 92 13 Check 100 25 1 yr. 5 da. air 48 25 48 100 48 100 14 Check 100 25 2 yrs. 5 da. air 40 25 40 100 40 100 254 Table 3. Series A— Boone County White -Continued Corn in a 75% Saturated Soil No. of Kerosene After No. No. Per ct. Per ct. Trial grains treatment treatment germ. in- j nred germ. norm, growth 1 50 dipped 3 da. air 27 54 54 50 1 min. 7 1 30 1 60 58 50 5 min. ; ) 26 1 52 50 Check 25 24 1 96 92 2 50 10 min. 3 da. air 28 1 56 54 50 15 min. } > 33 1 66 64 50 30 min. > > 30 60 60 Check 25 23 2 92 84 3 50 1 hr. 3 da. air 31 62 62 50 2 hr. } > 28 2 56 52 50 3 hr. >i 25 1 50 48 50 6 hr. > ) 22 1 44 42 50 8 hr. > » 23 46 46 50 14 hr. J 7 27 5 54 44 Check 50 45 1 90 88 4 50 1 da. 3 da. air 25 2 50 46 50 3 da. ; ; 30 6 60 48 50 6 da. > } 22 2 44 40 Check 25 23 1 96 88 5 100 10 da. 5 da. air 42 3 42 39 Check 25 24 96 96 6 100 25 da. 5 da. air 40 4 40 36 Check 25 25 100 100 7 100 30 da. 5 da. air 41 2 41 39 Check 25 23 92 92 8 100 50 da. 5 da. air 35 1 35 34 Check 25 25 1 100 96 9 100 120 da. 5 da. air 28 2 28 26 Check 25 25 100 100 10 100 158 da. 5 da. air 22 2 22 20 Check 25 24 96 96 11 100 190 da. 5 da. air 25 4 25 21 Check 25 23 92 92 12 100 215 da. 5 da. air 26 5 26 21 Check 25 25 2 100 92 13 100 1 yr. 5 da. air 12 4 12 8 Check 25 24 96 96 14 100 2 yr. 5 da. air 8 2 8 6 Check 25 25 1 100 96 255 2. LONG PEEIODS OF IMMERSION Reference has already been made to the work started on this problem in the laboratory of plant physiology at the University of Illinois previous to the publication of the report by Professor Forbes ('08). This work was abandoned before any definite results had been obtained, but the corn immersed in kerosene at that time (Feb- ruary 6, 1906) was set aside and kept in the storeroom of the labora- tory in a loosely covered fruit- jar until the present work was begun. The oil had become yellow and was of the consistency of thin syrup. There was about a pint of this corn — an amount far too small to per- mit any elaborate tests, but sufficient to demonstrate conclusively that under optimum conditions a considerable portion of it was capable of germination and perfectly normal growth. The majority of the trials recorded in Table 4 were made when the preliminary experiments, already referred to, were in progress. Of these trials, No. 3 yielded the highest per cent, of germination and was in every way the most satisfactory of any which had been made up to that time. After the treatment indicated in the table, the grains used in this trial were placed on filter-paper in a germinating pan with barely enough moisture present to initiate the growth proc- esses. As soon as a definite growth of root and coleoptile appeared the grains were transferred to soil in which the moisture was some- what higher but which did not exceed 30% saturation. This method was followed in all subsequent trials made with this corn. The seed- lings recorded in the column under per cent, of normal growth were just as vigorous and .had just as good color as the check seedlings which were grown from corn less than one year old. Plate XVI is a picture of two stalks of the corn grown from grains immersed in kero- sene for eight years (Trial 9, Table 4). A number of attempts were made to germinate grains of this corn in 50 and 75% saturated soil but all were complete failures. 256 Series A — Concluded Table 4. Golden Eagle Corn, immersed in kerosene February 6, 1906. Trial 1 was made July 11, 1911. The others followed as the time of the kerosene treatment indicates. Soil 30% saturated. Trial No. grains Kerosene treatment After- treatment Per ct. germ. % Norm, growth 1 50 5 yrs, 5 mos., 5 da. 48 hrs. clay, 12 hrs. air 40 40 2 50 5 J 48 hrs. plaster of Paris, 12 hrs. air 44 44 3 100 5 yrs., 5 mos., 22 da. 7 da. plaster of Paris, 1 da. air, 8 hrs. running water 64 55 4 35 •j > 60 da. plaster of Paris 65 56 5 35 )) None 32 32 6 25 5 yrs., 6 mos., 8 da. 60 da. plaster of Paris 58 50 7 25 }} 60 da. air 44 36 8 20 6 yrs., 2 mos. 10 da. air 50 40 9 20 8 yrs. 5 min. chloroform, 5 da. air 20 15 3. TYPES OF ABNOBMALITIES In soils of 50 and 75% saturation, abnormally swollen grains oc- curred frequently. A watery fluid collected inside the membranes in considerable quantities. By slight pressure several drops could be se- cured from a single grain. Microscopical examination of the ex- tracted liquid showed the presence of both perfect and corroded starch grains. Occasionally in the 75% saturated soil this liquid seemed to undergo fermentation. The pericarp, in these instances, was ruptured and the accumulated liquid made its way to the surface of the soil where it spread out and, drying, formed a hard white crust. Exami- nation of this crust under the microscope showed corroded starch grains and a large number of bacteria. The bacteria were of uniform shape and size and apparently belonged to a single species. Swellings as above described occurred among the grains which had failed to germinate and among those which were growing normally. They were also occasionally found among the normal grains used as a check, but much less frequently than among the treated grains. From the observations made, there was nothing to indicate that the swollen con- dition had any bearing on the germination or growth of the corn. 257 When punctured by a pin-prick the liquid inside the grain oozed out and no further accumulation of it occurred. This showed that it prob- ably was due to the high osmotic pressure inside the intact membranes and to an abundance of available water in the surrounding soil. No swollen grains appeared in cultures in which the moisture content of the soil was but 30% of saturation or less. One of the most noticeable injuries, though by no means the most frequent, was a curled and twisted condition of the leaves due to their inability to unfold normally in the process of growth. An examination of the tips of these leaves showed, in the majority of cases, that they were dead and that they adhered to each other on that account. It was possible to produce typical cases of the injury on control seedlings by touching the tip of the growing shoot immediately after it appeared through the coleoptile with an injurious reagent. Of the reagents used for this purpose sulphuric acid was the most certain to cause the abnormal growth. Kerosene applied in the same manner produced the injury, but it was by no means as effective as the sulphuric acid. The injury appeared occasionally among control seedlings, but there can be no doubt that the unusual frequency of the deformity in treated grains was due to the effects of the kerosene. Another abnormality attributable to the kerosene was a much enlarged and thickened coleoptile which the growing plumule occa- sionally failed to rupture. Whenever this unusual development ap- peared it was observed that the plumule had not grown nearly as far in the coleoptile as it ordinarily does. In some instances the plumule failed to develop, leaving the coleoptile entirely empty. This seemed to in- dicate that the coleoptile is less sensitive to the kerosene than the en- closed structures are, and that the enlargement is correlated with the failure of the plumule to develop. The most frequent injury was the death of the shoot. This oc- curred many times in grains from which the root grew normally. Very rarely in these experiments did the shoot grow when the root had been killed. The injuries mentioned above were not as distinct from each other as the descriptions might seem to indicate. As a matter of fact there was an imperceptible gradation from one to another. The de- formed leaves seem to represent the first visible injurious effects of the kerosene treatment. Increasing ill effects, due to an increase in the period of immersion, could be followed through a gradually decreasing vitality, to death. The action of the kerosene in producing injuries, and other evidences to be presented later, indicate that kerosene is not a violent poison to the growing corn-seedling. 258 4. DEY MEMBEANES It is evident that the kerosene did not act uniformly on the grains of corn which were subjected to the treatment. Some were killed, some injured, while others showed no injurious effects whatsoever. These conditions prevailed regardless of the period of immersion. No length of treatment was found which directly killed or even injured all the grains. This fact becomes significant when it is noted that the kerosene treatment was varied from a mere dipping to continuous im- mersion for a period of eight years. Rather early in my work it was suspected that the oil penetrated the membranes of some grains more readily than those of others. Some embryos had an oil-soaked appearance after the kerosene treat- ment while others seemed free from the oil. To obtain further evidence of the permeability of the coats to the oil, 200 grains of Champion White Pearl were placed in kerosene colored with Sudan III. After 50 days' immersion the corn was re- moved and superficially dried with a towel. One hundred of these grains, taken at random, were cut transversely through the middle of the embryos and carefully examined for the presence of colored oil. Seventy-eight showed no trace of oil or color in the embryos; five were slightly stained; the remaining seventeen were deeply stained and showed the presence of oil in considerable quantities. In no case was there any evidence of oil in the endosperm. The remaining 100 grains treated with Sudan III kerosene, as above indicated, were left exposed to the air for twelve hours and then planted in a 30% satu- rated soil. In cutting through the 100 grains taken at random from the 200 treated, it very soon became apparent that in most cases a selection from external appearances alone could be made. This was attempted before planting the remaining 100 of the treated grains. From external examination these 100 grains were divided into three groups : first, those seeming to be free from the colored oil ; second, those showing slight traces of it; and third, those in which the em- bryos were deeply stained. The grains of these groups were planted in separate rows in the culture pans and were kept under identical con- ditions. Of the 76 grains of group one, all germinated and produced normal seedlings. Nine out of 14 of the second group, germinated but produced seedlings showing greater or less injury. One grain from group three germinated, the seedling being decidedly weak. It has already been stated that the grains were carefully selected before they were immersed in the oil. Any having visible defects were rejected, but no selection was made after removing the corn from the oil,- with the following exception : April 20, a quantity of the 259 Champion White Pearl was removed from the kerosene in which it had been placed February 6 — 76 days' immersion — and 50 grains were selected from it which seemed from external appearances to be free from kerosene. These grains were exposed to the air for ten days and then planted in a 30% saturated soil. Forty-nine, or 98% of them, developed normal seedlings. From these results it is apparent that an almost perfect germination can be secured by selecting grains showing no traces of stain in the embryos. The selection of grains with membranes slightly permeable to the colored oil can by no means so easily be made. The structures at the tips of the grains always take up the oil readily and it spreads for some distance from them, giving the appearance of stain within the embryos when in reality it is en- tirely superficial. The absence of Sudan III in the grain does not necessarily mean the absence of kerosene, since the membranes may be semi-permeable. A number of experiments were undertaken to test this assumption. Grains immersed in Sudan III kerosene for long periods and free from stain were carefully dissected, and the structures within the coats were tested by the picric acid methods of Schulz ('08), and Krauz ('09). The results were uniformly negative. It was found that tests by these reagents were not nearly so delicate as the sense of taste. In no case, however, could the presence of kerosene be de- tected in unstained grains. On the other hand, it could be readily de- tected in grains which had been but slightly stained with Sudan III. It should be here stated that the above holds true only for air-dry grains. These facts indicate very clearly that the kerosene enters some of the grains and is excluded from others. Whether the membranes of the grains showing penetration had been mechanically injured or were of different physical structure has not been determined. In either case the result would be the same. Undoubtedly there are many opportunities for mechanical injuries, but the fact that the number of grains exhibiting a penetration of the kerosene increases with the time of immersion would indicate that the membranes are not uni- formly impermeable. Membranes of widely different properties are not uncommon in seeds of the same kind. Many cases of delayed germination are at- tributed to this peculiarity [see Crocker ('06) ; Hanlein ('8o) ; Nobbe and Hanlein ('77)]. It is not unlikely that the membranes of the corn kernel are sufficiently different in their organization or development to permit a rather wide variation in their permeability to kerosene. A number of interesting studies on the physical properties of plant membranes have appeared recently. Brown ('07 and '09) found 260 the "seed" of Hordeum vulgare to be enclosed in a semi-permeable membrane. He found the aleurone layer of Hordeum vulgare to con- tain a pigment which serves as an indicator for acids and alkalis. This was not only a very interesting discovery but one which materially aided in the successful conduct of his work. He learned that the in- tact membranes of H. vulgare are impermeable to sulphuric acid; con- sequently when, in the presence of this acid, the purple pigment changed to a pink color it indicated imperfect membranes. Thus it was possible for him to select "seed" with intact membranes for ex- perimental purposes. From all indications, Sudan III is just as ef- ficient for determining imperfections in the membranes of Zea as are the color reactions described by Brown. Schroder ('n), using Brown's methods, found the same kind of semi-permeable membranes in wheat. More recently Shull ('13), has made similar studies on the tests of Xanthium glabratum and demonstrated selective semi-permeability like that found in Hordeum. 5. MUTILATED MEMBEANES To determine the toxic action of kerosene on the embryo, the outer membranes were punctured at several places and also removed. The following tables (Series B, tables 5 to 10 inclusive) give the re- sults. Series B Tables 5 to 10 inclusive. Effects of kerosene on grains with mem- branes punctured before immersion. Champion White Pearl Corn germinated in a 25% saturated soil. Table 5. Normal Grains (Control) Trial No. of Kerosene After No. No. in- Per ct. fc Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 100 100 2 50 10 days ;; 50 100 100 3 50 15 days >) 50 2 100 96 4 50 20 days n 50 2 100 96 5 50 25 days }} 46 3 92 86 6 50 35 days }> 44 6 88 76 7 50 50 days )> 40 4 80 72 8 50 75 days }) 38 1 76 74 Table 6. Pedicle Eemoved No. of Kerosene After No. No. in- Per ct. % Norm. Trial grains treatment treatment 3 da. air germ. j ured germ. growth 1 50 5 days 50 100 100 2 50 10 days ) > 50 100 100 3 50 15 days n 50 100 100 4. 50 20 days ; 1 50 1 100 98 5 50 25 days .*> 50 100 100 6 50 35 days ;> 50 100 96 7 50 50 days ' ■' 50 1 100 98 8 50 75 days t) 47 94 94 261 Series B — Concluded Table 7. Pericarp Punctured at Distal End of Coleoptile Trial No. of Kerosene After No. No. in- Per et. % Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 30 300 40* o 50 10 days >> 44 38 88 32 3 50 15 days ft 34 32 68 04t 4 50 20 days S 1 30 28 60 04 1 5 50 25 days j? 22 22 44 00 6 50 35 days > } 6 5 12 02 7 50 50 days it 00 00 8 50 75 days )) 1 1 02 00 *Not so vigorous as control. tRetarded and decidedly weak. Table 8. Membranes Lying within the Pedicle Punctured Trial No. of Kerosene After No. No. in- Per ct. % Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 100 100* 2 50 10 days }} 50 100 100* 3 50 15 days > i 48 40 96 16t 4 50 20 days ; J 50 42 100 16 1 5 50 25 days >i 32 32 64 00 6 50 35 days 17 30 28 60 04 7 50 50 days it 20 20 40 00 8 50 75 days it 00 00 *Slightly retarded. tRetarded and weak. Table 9. Pericarp Eemoved Trial No. of Kerosene After No. No. in- Per ct. %Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 12 100 76* 2 50 10 days it 50 10 100 80* 3 50 15 days )) 36 20 72 32* 4 50 20 days 1 7 30 30 60 00 5 50 25 days 7) 16 16 32 00 6 50 35 days i ; 10 10 20 00 7 50 50 days 17 2 2 04 00 8 50 75 days if 00 00 ♦Retarded and weaker than control. Table 10. Pericarp Eemoved Trial No. of grains 1 50 2 50 3 50 4 50 5 50 6 50 7 50 8 50 Exposed to dry air 5 days 10 days 15 days 20 days 25 days 35 days 50 days 75 days No. No. in- Per ct. %Norm. germ. jured germ. growth 50 5 100 90 48 7 96 82 46 ?* 92 ?* 45 ?* 90 ?* 46 ?* 92 ?* 40 ?* 80 ?* 48 ?* 96 ?* 46 ?* 92 ?* *These seedlings were uniformly weak. It could not be told at the time of observa tion whether they would recover or not. 262 The fact that the pericarp is greatly modified at the tip of the grain — the pedicle — into a very porous vascular tissue, introduces a factor that greatly increases the difficulty in a study of the mem- branes. At this point, however, the pericarp is reinforced within by a compact remnant of the nucellus which, when perfect, effectively pre- vents the oil from penetrating the grains. There can be no question but that this is the usual point of ingress into those grains in which the embryos are stained. The colored oil invariably makes its appearance here and gradually passes up through the embryo. That the pedicle itself is not only valueless for excluding the oil but that it is the source of positive injury is shown in Table 6. The pedicles were carefully broken off from these grains before immersing them in kerosene, and it was found that a higher rate of germination resulted from grains so treated. It is altogether likely that the spongy tissue of the pedicle, by retaining rather large quantities of the kerosene, is responsible for a decrease in germination when it is not removed. The oil remaining in or on the grains seems to be absorbed and carried to the regions of growth as soon as the growth processes are initiated. If the amount is beyond a certain limit, injury is produced. The -effects of removing the pedicle and slightly puncturing the membranes within it are shown in Table 8. The best place to puncture the pericarp without injuring the embryo is near the distal end of the coleoptile where, in the process of maturing, a small wrinkle is formed in a rather large proportion of the grains. The membranes at this point can readily be ruptured with a needle without the slightest in- jury to the underlying parts. The effects of thus puncturing the coats (Table j) are in a gen- eral way comparable to those secured by puncturing the membranes lying within the pedicle. In the latter case, however, the grains did not show the injurious effects as quickly as in the former. In both cases the colored oil penetrated the embryos in sufficient quantities to be plainly visible in twenty-four hours. It is important to note that, excepting a slight retardation, ioo% of the grains in Table 8 con- tinued to produce normal seedlings after immersion in kerosene for a period of ten days. This shows that the presence of a limited amount of oil in the embryo is not necessarily injurious. By soaking the grains in tepid water for ten minutes the entire pericarp, including the pedicle, is very easily removed without in- jury to the parts within. After thus removing it, the grains were dried at room temperature for five days and then immersed in kero- sene. The results (Table 9) correspond closely to those obtained in the experiments with punctured membranes and show that a punctured 263 ^•■'V'.V I >■■.. ■::,■■■::*,'■:■■£.■- pericarp is -equivalent to its removal. In either case the dormant grains are killed within a comparatively short time (75 days). It will be noticed that grains with the pericarp removed but not otherwise treated (Table 10) retained the power of germination to a fairly high degree for the time indicated in the table ; but such grains after ten days' exposure to the dry air of a steam- heated room produced seedlings that were uni- formly weak. This indicates that these mem- branes play a very important role in preserving the vitality of the grains. The dry pericarp, not including the pedicle, was shown to be impermeable to kerosene in an- other way. A large grain of the Champion White Pearl furnishes a membrane fully one-half inch in diameter. It is easily removed after soaking the grain for a few minutes in warm water. After drying, it can be cemented over the end of a glass tube for use either as a barometer or as an osmometer. This simple piece of apparatus was, as far as I know, first devised by Becquerel ('07) in his studies on the permeability of seed coats to certain gases. Shull ('13) also used it with success in demonstrating the semi-permea- bility of the testa of Xanthium. Adapted for my work, the apparatus was constructed as shown in the accompanying sketch. Considerable difficulty was experienced in finding a cement not soluble in kerosene. Sealing-wax, such as is used by express com- panies for sealing valuable packages, was finally found to serve the purpose admirably. The rubber stopper at the end of the glass cylin- der serves as a foundation to which the membrane is cemented. The small glass tube (Fig. 1, cc) was allowed to protrude three or four millimeters through the perforated rubber stopper. A layer of wax equal in thickness to th-e protruding portion of the tube was then ap- plied and the edges of the membrane were pressed into it while it was still soft. More wax was then applied to make the seal perfect. The central portion of the membrane over the end of the small glass tube was left entirely free from wax. Such an apparatus was set up March 7, with kerosene on one side of the membrane and with plaster of Paris as an absorbent on the other. At the present time (May 6) the wax is holding perfectly and there has been no trace of oil passed through the membrane. A Fig. 1. Apparatus used in testing directly the per- meability of the pericarp of Zea: a, membrane; bb, rubber stopper; cc, glass tube; d, receptacle for liquid to be tested. 264 similar apparatus was set up as a barometer November 29 and has supported a mercury column representing a complete atmospheric pres- sure since that time (5 mos., 7 days). The mercury rises and falls with the changes in atmospheric conditions, but no fall attributable to the penetration of air through the membrane has taken place. No effort has been made to extend these studies beyond the limits indicated in the title of the paper, but as a matter of interest the ap- paratus was set up as an osmometer with a saturated solution of sodium chloride on the inside of the membrane and distilled water on the outside. The contents of the upper tube, a cross-section of which had the same area as the exposed membrane, rose at the rate of 4^ cm. a day for four days. Before the rise had ceased the liquid out- side the membrane was tested with silver nitrate for the presence of sodium chlorid'e. The test showed the presence of the salt in large quantities. From these experiments it may be concluded that under the con- ditions described the membrane is impermeable to kerosene and to atmospheric gases, but that it is permeable to sodium chloride. 6. MOISTUEE CONTENT OF THE GRAINS The data thus far discussed pertain to the corn which was thoroughly air-dried before it was immersed in kerosene. The fol- lowing experiments show the effects of similar treatments on grains containing different amounts of moisture at the time of immersion. Series C Tables 11 to 14 inclusive. The effects of different amounts of water in the grains at time of immersion in kerosene. Champion White Pearl Com germinated in a 25% saturated soil. Table 11. Water in Grains Desiccated to Constant Weight at 100° C. Samples (50 grains) Original weight Dry weight Ratio of water to dry weight Old corn 24.040 26.832 28.185 23.523 22.112 22.468 2.19% 21.30% 25.44% New corn New corn soaked 1 hr Table 12. Old Corn. Water Content Equivalent to 2.19 per cent, of Dry Weight Trial No. of Kerosene After No. No. in- Per ct. % Norm. grams treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 100 100 2 50 ■ 10 days } ) 50 100 100 3 50 15 days ; ! 48 4 96 88 4 50 20 days 1 > 46 92 88 265 Series C — Concluded Table 13. New Corn. Water Content Equivalent to 21.3 per cent, of Dry Weight Trial No. of Kerosene After No. No. in- Per ct. % Norm. grams treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 46 4 92 84 2 50 10 days > > 36 8 72 56 3 50 15 days ; ? 8 8 16 00 4 50 20 days ; ? 00 00 Table 14. New Corn Soaked in Water 1 hr. Water Content Equivalent to 25.44 per cent, of Dry Weight Trial No. of grains Kerosene treatment After treatment No. germ. No. in- jured Per ct. germ. % Norm, growth 1 2 3 50 50 50 5 days 10 days 15 days 3 da. air 42 10 1 5 84 20 00 82 10 00 The amount of water contained in the respective samples used, as shown by desiccation to constant weight at ioo degrees C, is re- corded in Table n. The old corn was harvested one year previous and had been stored in the dry rooms of the laboratory for approxi- mately six months. The new corn had just been harvested, but was fully mature and sound in every way. It will be noted that the old corn contained an amount of water equivalent to 2.19% of the con- stant weight at 100 degrees C. ; the new corn, 21.3%; and the new corn soaked in water for one hour, 25.44%. Under normal condi- tions both the old corn and the new corn germinated perfectly. The corn containing water in the amounts indicated above was immersed in kerosene and tested for viability at intervals of five days. The results are brought together in tables 12, 13, and 14. A glance at these tables shows that after a period of more than five daA^s' immer- sion in kerosene the injuries are very decidedly increased in the grains of high water content. One hundred per cent, of the dry grains germi- nated after ten days' immersion in kerosene, while the germination of the new corn and the new corn soaked in water dropped to 36 and 10% respectively. Immersion in kerosene for twenty days proved fatal to all the corn of each lot containing the higher percentages of water. The air-dry corn, after an equal period of immersion, gave 92% germination and 88% normal growth. Since the per cent, of germination falls so rapidly in corn not fully dry and in corn soaked in water for a short time, it seems evident that some physical change of the investing membranes takes place on moistening, and that they become more readily permeable to kerosene. 266 After twenty days' immersion in Sudan III kerosene, and after germination tests had proven all the grains dead, the remaining grains of the new corn were carefully examined to determine the number showing penetration of the stain. Of the 161 grains, 24, or approxi- mately 15%, were stained. Twenty-four days later — 74 days' immer- sion — the number had increased to 45, or 25%. If one should attempt to judge the viability of the grains by the presence or absence of the stain, as was done so effectively in the dry grains, the rate of germina- tion should be approximately 75%. Both stained and unstained grains, however, had lost all power of germination and the presence of kerosene was easify demonstrated in both. The conclusion nat- urally follows that the membranes of the moist grains permit the penetration of the kerosene, but that they effectively prevent the pas- sage of the Sudan III. The percentage of grains stained by Sudan III was approximately the same as in the dry grains. This supports the view previously expressed; namely, that the stained embryo is an in- dication of imperfect membranes. 7. VARIATIONS IN SOIL MOISTURE That some grains of corn bear immersion for a period of eight years in kerosene is experimentally proven. This, however, is not true of all grains of like origin subjected to similar treatment. In every sample taken at random a certain percentage of the grains fail to germinate after a comparatively short period of immersion. By means of the Sudan III it has been conclusively established that a limited number of grains of a random sample are stained and that these even- tually fail to germinate even under the most favorable conditions. Death in these instances is due to the toxic action of the kerosene on the dormant embryo. Since it has been shown that the dry mem- branes are impermeable or only slightly permeable to kerosene, the presence of the oil within the membranes, in sufficient quantities to cause death, is attributable to imperfect membranes. The presence of small quantities of kerosene within the grain, however, does not neces- sarily prove injurious. Grains immersed in kerosene for the same periods of time give very unlike results when placed under different conditions for germination. It was found that in the presence of abundant moisture the injurious effects of the kerosene treatment are especially marked. In the experiments (Series A) in which grains, similarly treated with kerosene, were placed in soils with different moisture content, this injury was clearly brought out. When the amount of water in the soil was reduced from 30% saturation (Series A, Table 1) to 25% saturation (Series B, Table 5) the per cent, of 267 germination was increased and the growth of the seedlings was more nearly normal; but when the water content of the soil was increased to 50 or 75% of saturation (Series A, Tables 2 and 3) the per cent, of germination was markedly decreased and the subsequent growth of many of the seedlings abnormal. The germination and growth of grains immersed in Sudan III kerosene but unstained is normal in 25 and 30% saturated soils. The slightly stained grains, that is those containing small quantities of kerosene, frequently produced normal seedlings when the water con- tent of the soil did not exceed 25% saturation. In 30% saturated soil the per cent, of normal growth of these seedlings was greatly re- duced. In soils of 50 and 75% saturation all grains showing the slightest penetration were killed, as were also a considerable number in which the presence of oil could not be detected from external ex- amination. Traces of kerosene are always present when once the grains have been immersed in it. This is shown by the decreased germination in soils of high water content and also by other and more direct evi- dences. Grains immersed for comparatively short periods retain the taste of the oil after six months' exposure to dry air at room tempera- ture. Because of the varying moisture content of the corn, and possibly changes due to the presence of the kerosene, the exact amount of oil taken up and retained could not be accurately determined. Quantita- tive evidence, though desirable, was not necessary to show that a con- siderable residue remained after volatilization had been carried to the limit used in this work. The question, then, of the disposition of the oil or its residues in those cases in which no injurious effects are pro- duced becomes important. Schmidt ('91), in his studies on the translocation of oils in the living plant, devised a method by which he succeeded in directly intro- ducing almond oil, cocoa butter, and other oils into the tissues of the stem. He showed that these oils were taken up and moved with con- siderable rapidity through both stem and leaf. He concluded that both neutral oils and fatty acids could be taken up by the growing plant, saponified and emulsified in a manner similar to that carried on in the animal organism. Kryz ('09 and '13), investigating the effects on plants of oils used as insecticides, treated Impatiens with vaseline, and Datura and Alisma with kerosene. In the latter case he planted the seeds in flower-pots containing garden soil and sprinkled the soil with a 5% solution of the oil both before germination and after the plants had reached considerable size. He showed that the oil was taken up 268 and carried through the vascular tissues to the leaves, where it was stored in quantities sufficiently large to make its presence easily deter- minable. Unfortunately Kryz continued the treatment until the plants were killed. He seems not to* have paid any attention to the power of recovery of the plant from injuries not at once fatal. These investigations led me to believe that under favorable con- ditions a limited amount of kerosene might be absorbed and disposed of, without injury, by the growing corn seedling. Observations con- firmed this belief. The coleoptiles of seedlings grown from grains immersed in colored oil frequently showed the red stain. In soils of low moisture content these seedlings developed normally, while in soils of high moisture content they were either killed or showed pronounced injury. Numerous attempts were made to demonstrate the presence of the oil in the tissues. Sections were treated with Sudan IlT, alkannin, and picric acid benzol (5), but because of the large amount of oil normally present in the structures of the young corn seedling and the very small amount of kerosene which ordinarily is present, the results were not successful. No satisfactory test for demonstrat- ing the presence of kerosene in very small quantities has been found. The experiments of Kryz were repeated in a modified form and his results confirmed. Corn seedlings were grown on filter-paper so that the roots penetrated the paper and entered soil contained in a pot below. When the seedlings were about three inches tall, from 1 to 3 drops of Sudan III kerosene were applied to the old grains at the base of the seedlings. A drop was equal to one-fiftieth cubic centimeter. In a few minutes the stain showed prominently in the stems of the seedlings and eventually reached the leaves in quantities sufficiently large to be plainly visible to the naked eye. All the seedlings treated with three drops died within five days after the treatment. The majority of the seedlings treated with one and two drops recovered. The amount of oil disposed of was certainly many times as much as could be retained in the dry grains immersed in oil and afterwards treated to eliminate it. It is apparent that the older seedlings can dis- pose of a much greater amount of oil than the younger ones. It is evident that within certain limits the seedlings are not in- jured by the oil present at the time of planting provided growth is initiated in the presence of a minimum amount of water. The small quantities of kerosene are toxic in proportion to the increase of the moisture content of the soil. In the 50 and 75% saturated soils the dormant period of the grain is always less than 36 hours, while in a 25% saturation the time is extended to approximately five days. This increase of time affords the seedling an opportunity to dispose of the oils much more slowly, and it does so without injurious effects. 269 8. OTHER OILS In addition to the kerosene the effects of a number of other petroleum oils have been studied. At the present time only the in- itial results have been obtained. These results indicate that the in- juries due to the penetration of the dormant grains by the oils are 'essentially the same as in the case of kerosene. The effects on the germinating grains, however, differ very widely. The more volatile oil (gasoline, Table 16) produces no more injury than does the kero- sene. From present indications it seems probable that a high moisture content of the soil affects the grains immersed in the more volatile oils less than those immersed in kerosene. On the other hand, the injuri- ous effects of the heavier oils on germinating grains in soils of either low or high moisture content are much more pronounced. The same means were employed for eliminating these oils from the grains after immersion as were used with the kerosene; viz., wiping the grains carefully with a towel and then exposing them to the air. The heavier oils do not volatilize as completely as the kerosene and gasoline do. The residues dry on the grains, producing a hard coating which pre- vents normal germination. At present the trend of evidence tends to show that the grains bear immersion in the lighter oils without injury for much longer peri- ods than in the heavier oils, and that the injurious after-effects of the latter are more pronounced than those of the former. SERIES D Tables 15 to 20 inclusive. Comparisons between kerosene and other petroleum oils. Champion White Pearl Corn germinated in a 25% saturated soil. Table 15. Kerosene (Control) Trial No. of Kerosene After No. No. in- Per ct. Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 50 100 100 2 50 10 days ; j 50 3 100 94 3 50 15 days 7 J 47 3 94 88 4 50 20 days ; 5 47 4 94 86 5 50 25 days ?> 45 1 90 88 6 50 35 days ' ' 43 1 86 82 7 50 50 days 7 > 39 2 78 74 8 50 75 days 34 1 68 66 Table 16. Gasoline Trial No. of Gasoline After No. No. in- Per ct. % Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 49 1 98 96 2 50 10 days } } 49 3 98 92 3 50 15 days >t 47 2 94 90 4 50 20 days 7 7 47 3 94 88 5 50 25 days >t 45 1 90 88 6 50 35 days 7 > 42 1 84 82 7 50 50 days )> 40 80 80 8 50 75 days 7 7 37 1 74 72 270 Series D — Concluded Table 17. Kansas Crude Oil JNo. of Oil After JNo. No. in- Per ct. % Norm. Trial grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 46 2 92 88 2 50 10 days >> 48 2 96 92 3 50 15 days it 50 6 100 88 4 50 20 days it 47 3 94 88 5 50 25 days >i 43 4 86 78 6 50 35 days it 44 5 88 78 7 50 50 days >j 50 20 100 60 8 50 75 days t> 40 10 80 60 Table 18. Heavy Eed Oil No. of Oil After JNo. No. in- Per ct. %Norm. Trial grains treatment treatment germ. jured germ. growth -> 50 5 days 3 da. air 50 100 100* 2 50 10 days )) 46 92 92* 3 50 15 days !) 40 5 80 70* 4 50 20 days ft 40 4 88 76* 5 50 25 days 40 «J 80 74* 6 50 35 days It 48 4 96 88* 7 50 50 days } ; 40 28 80 24* 8 50 75 days it 24 10 48 28* *A11 seedling's decidedly retarded. Table 19. Fuel Oil No. of Oil After No. No. in- Per ct. % Norm. Trial grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 48 2 96 92* 2 50 10 days 46 14 92 64* 3 50 15 days 46 18 92 56* 4 50 20 days 42 15 84 54* 5 50 25 days 44 14 88 60* 6 50 35 days 48 b 96 80* 7 50 50 days 42 26 84 32* 8 50 75 days 40 20 80 40* *From 2 to 3 days retarded. Table 20. Engine Oil Trial No. of Oil After No. No. in- Per ct. %Norm. grains treatment treatment germ. jured germ. growth 1 50 5 days 3 da. air 48 2 96 92* 2 50 10 days 48 20 96 56* 3 50 15 days 48 18 96 60* 4 50 20 days 45 16 90 58* 5 50 25 days 43 15 86 56* 6 50 35 days 40 12 80 56* 7 50 50 days 44 32 88 24* 8 50 75 days >> 40 24 80 32* ♦Retarded 3 days and very uneven. 271 9. SUMMAEY OF CONCLUSIONS Grains of Zea mais may be immersed in kerosene for periods of ten to twenty days without injury if the optimum conditions for the germination and growth of such grains are provided. These condi- tions include the removal of the superficial oil from the grains and the presence of a minimum amount of water during germination and in- itial growth. Injuries which occur to the dry grains immersed in kerosene for longer periods than above indicated are due to the penetration of the oil into the embryos through imperfect membranes. The dry membranes covering the corn embryo, when perfect, are impermeable to kerosene and to Sudan III. Some grains of Zea mais may be immersed in kerosene for eight years without injury to the dormant embryo. The life of dormant grains, with membranes which have been mechanically injured, is destroyed within seventy-five days after im- mersion in kerosene. Kerosene is injurious to the germinating grains in direct propor- tion to the length of time of immersion and to the increase of the- water content of the soil above the minimum required for germina- tion. When moist grains are immersed in a solution of kerosene and Sudan III, the membranes are penetrated by the kerosene but not by the Sudan III. The membranes are, therefore, semi-permeable. The germinating corn grain may absorb and dispose of a limited amount of kerosene without injury. The smaller the amount of water present during germination the larger the quantity of kerosene which can be disposed of. Older corn seedlings may dispose of compara- tively large quantities of kerosene without injury. It is not advisable to treat seed corn with kerosene unless the water content of the soil is under control. The injurious effects of petroleum oils on germinating corn seem to vary inversely as the volatility of the respective oils. 272 BIBLIOGRAPHY Becquerel, Paul. '07. Recherches sur la Vie Latente des Graines. Ann. Sci. Nat., Bot, 9 e ser., 5 : 193. Brown, Adrian J. '07. On the Existence of a Semi -permeable Membrane enclosing the Seeds of some of the Gramineae. Ann. Bot., 21 : 79. '09. The Selective Permeability of the Coverings of the Seeds of Hordeum vulgare. Proc. Roy. Soc. London, Ser. B, 81 : 82. Crocker, William. '06. Role of Seed Coats in Delayed Germination. Bot. Gaz., 42 : 265. Duggar, B. M., and McCool, M. M. '09. Suggestions concerning Treatment of Seed Corn with De- terrents against Crows. Circular No. 6, Cornell Agr. Exper. Station. Forbes, S. A. '08. Experiments with Repellents against the Corn Root-aphis, 1905 and 1906. Bull. No. 130, 111. Agr. Exper. Station. Hanlein, H. '80. Ueber die Keimkraft von Unkrautsamen. Landw. Ver- suchs-Stat, 25 : 465. Krauz, Cyrill. '09. Beitrag zur Schulzschen Farbenreaktion der Mineralole. Chem. Zeit., 33 : 409. • Kryz, Ferdinand. '09. Uber den Einfluss von Erdol auf die Entwicklung von Datura und Alisma. Zeit. f. Pflanzenkrank., 19: 449. '13. liber die Aufnahme von Vaselinol durch Balsaminen. Zeit. f. Pflanzenkrank., 23 : 34. Lummis, G. M. '03. Effect of Coal Tar, Coal Oil, Gasoline, Benzine and Kero- sene on Germination of Maize. Proc. 24th Ann. Meeting Soc. Promotion Agr. Sci., p. 96. 273 Nobbe, F., und Haenlein, H. 'yy. Ueber die Resistenz von Samen gegen die ausseren Factoren der Keimung. Landw. Versuchs-Stat., 25: 71. Schmidt, R. H. '91. Ueber Aufnahme und Verarbeitung von fetten Oelen durch Pflanzen. Flora, 74 : 300. Schroder, H. '11. tiber die Selective permeable Hiille des Weizenkornes. Flora, 102: 186. Schulz, Ferdinand. 'o8. Eine neue Reaktion der Mineralole. Chem. Zeit., 32 : 345 Shull, Chas. A. ' i-2- Semi-permeability of Seed Coats. Bot. Gaz., 56: 169. VITA 1873, Oct. 3. Born in Stark County, Illinois. 1879 — 1887. Attended country school in Penn township, Stark County, Illinois. 1887 — 1 89 1. Time divided between farm and country school, Stark County, Illinois. 1891 — 1892. Student in High School, Bradford, Illinois. 1892 — 1894. Teacher in country school, Stark County, Illinois. 1894 — 1896. Student in Illinois State Normal \Jniversity, Normal, Illinois. 1896 — 1897. Principal, Public Schools, Diamond, Illinois. 1897 — 1899. Student, Illinois State Normal University, Normal, Illinois. Graduated 1899. 1899 — 190 1. Principal, Public Schools, Golconda, Illinois. 1901 — 1903. Principal, Public Schools, St. Anne, Illinois. 1903 — 1910. Superintendent, Public Schools, Onarga, Illinois. 1910 — 1914. Assistant in Botany, University of Illinois. Degrees 191 1. A.B., University of Illinois. 1912. A.M., University of Illinois. Pl.ATK XVI LIBRARY OF CONGRESS i i nun 000 935 204 1