THE PERMEABILITY OF THE SEED COAT OF PEAS BY SIEGFRIED PAUL HARTER THESIS FOR THE DEGREE OF BACHELOR OF ARTS IN BOTANY COLLEGE OF LIBERAL ARTS AND SCIENCES UNIVERSITY OF ILLINOIS 1922 / 922 h)25 UNIVERSITY OF ILLINOIS Ma_£_ 25j_ THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Si e Lgfr i ed_ _Paul_ _Har te r ENTITLED T-he- 1-i ty - -of - -th-e - -S ee-d- -G ©-at- -q£- - P-e a© IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF Bachelor of Arts in Botany Instructor in Charge 4 HEAD OF DEPARTMENT OF __J?j)tany_ Digitized by the Internet Archive in 2015 https://archive.org/details/permeabilityofseOOhart 1 Table of Contents. I Introduction. II Citation of References. Ill Methods and Materials. IV Results and Experimental Data. V Discussion and Interpretation. VI Conclusions. VII Figures. 2 I Introduction. It has long been known that dry seeds, spores, and other dormant stages of plants possess an enormous resistance to very poisonous substances. The early investigations along this line were carried on principally with the weaker solu- tions. More recently a great deal of work has been done with the higher concentrations of acids and salt solutions, with especial reference to their effect upon the dry seeds of the leguminous plants. Many of these investigators have reported certain maxima of poisonous effect at certain concentrations of the toxic substances. One of the earliest of these exper- iments was carried on by V. Arcichovskij, and his results are set forth in an article in Biochem. Zeitschrift Vol. 50, 233, «< ft entitled "Biochemi sche wirkung hochst Konzentrierter Losungen". His results are especially interesting here because his work forms the basis for a considerable portion of this investigation. Arcichovskij carried on his work with the seeds of peas, but the particular variety of seeds which he used is not men- tioned. In the course of his experiments he treated seeds with Formalin, Sulfuric acid, and ITitric acid; using varying concentrations and a varying period off exposure. Elaborate precautions were taken against bacterial contamination of the germinating seeds and a very scrupulous system of washing the treated seeds was resorted to. The greater part of his work was carried on with formalin and he concluded for this poison that the stronger concentrations of formalin even after a long periods of immersion showed relatively small poisonous 3 effect, whereas concentrations of medium strength gave maxi- 2 mum killing. The highest poisonousness was evinced by the Q% formalin. In his tests with Sulfuric Acid he used a 2 hour imer- sion in all tests and varied the concentrations from n/128 to concentrated. The table of results, as he gives it, follows: Cone. H2S04 n/128 n/32 n/8 n/4 n/2 n/l 2n 4n 8n 16n 32n % of germ 94 92 76 48 24 49.5 24.5 92 100 100 100 Cone. 100 . As in the case of the work with formalin it will be observed that a higher percent of germination was obtained with the stronger concentrations of acids. Here a maximum killing was obtained at l/2n solution. He further found that some seeds will withstand a 16 hour storage in concentrated H2S04. With Nitric acid a maximum poi sonousness was obtained with a normal solution and a 2 hour treatment. The general conclusion from the work was that the resistance of seeds to poisons depends upon the physical properties of the solutions and that the strongest concentrations in all cases are the least poisonous. A more recent study of the same problem is that of A. J. Brown. This work was conducted on the seeds of Horedum vul- gare, and the results are reported in an article entitled n 0n the Existence of a Semipermeable Membrane enclosing the Seeds of some Gramineae. " Here too, various concentrations « * . - - - , . « 4 of H2S04 were used, together with various lengths of treat- ment. In the particular variety, caerulescens, which was used, the grain contains a blue pignent in the aleurone cells which turns to red with acid, thus making it a valuable in- dicator. Brown found a marked difference in the behavior of damaged seeds and those which were intact. The damaged seeds changed color at once in even a 1 $ solution of H2S04, whereas in the case of perfect seeds no color change resulted even after a week’s treatment in such a solution. In this case, however, swelling took place at once, thus indicating that water had entered the seed. Sections cut longitudinally and cross wise through such seeds also show no trace of the en- trance of acid, but give abundant evidence of the admission of water. By titrating acid solutions before and after steeping seeds in them it was also found that undamaged seeds abstract water from H2S04 solutions not exceeding 4*9$. In working with more concentrated solutions 9, 18, and 36$ H2S04 for 44 hour periods of treatment no acid was ab- sorbed in any case by the undamaged seeds, and the amount of water absorbed decreases inversely as the concentration of the acid. Good germination was secured, after washing, with treat- ment in 18 to 36$ solutions. The same selective action was manifested for HN03 and Iodine, although HN03 proved, by first boiling the undamaged grains and then treating with acid, that this semi-permeability is not a function of the living proto- plasm, but is resident in the seed coat. An attempt was also made to find the particular portion of the seed coat which ' , " , - 5 was responsible for the semi-perm eabl e property. Brown came to several very definite and significant con- clusions. He found that the grain of Hordeum, Avena, Triticum, and Secale, is enclosed in a semi-permeabl e covering which permits the passage of water and iodine to the interior of the grain, but prevents the passage of H2S04, HC1 , and all salts of metals thus far examined, when they are in aqueous solution. This semi -permeable or selective property is located someplace in the sperraoderm of the seed. It would appear that a large proportion of the work which has already been done in connection with the problem is more or less irrelevant to the real bearings of the question. Arcichovskij placed an undue emphasis upon uselessly elabor- ate washing and sterilization methods, whereas he failed to be rigorous enough in his germination criterion. Many seeds which he would count as germinated are in reality damaged be- yond viability. His general conclusions that the poisons affected the germination of the seeds to a greater or lesser extent, and that thereby resistance to microorgainisms was weakened, fail to give any explanation for the difficulty. He admits that the reasons for the lessened poisonous effect of higher con- centrations are probably very complex. It is also quite apparent that the dry protoplasmic state of the seeds causes them to be particularly resistant, and that the chemical ac- tivity of substances in higher concentrations is much less than in weaker ones. Like Kahlenberg and True, Arcichovskij * 6 lays stress on the fact that in the case of electrolytes, stronger solutions are relatively and absolutely less disso- ciated. H. Schroeder, on the other hand, in his article in Slora ii Vol. 102, p 186, "liber die Selektiv permeabele Hulle des Weizenkornes" concludes that peas do not possess a selective permeable membrane. He finds that peas manifest no difference in absorption rate when immersed in increasing concentrations, nor is there any difference in absorption rate between injured and uninjured seeds. He points out that any change in the con- centration of the surrounding solution in the case of pea seeds is of only temporary significance. In a Cornell University bulletin entitled " Germination of Seed as affected by Sulfuric Acid Treatment", Love and Leighty attempt to make a practical use of the results of experimental investigations of the cause of hard seeds. In using varieties of clover, alfalfa, sweet clover, weed seeds, and cotton seeds, they got results which were too inconsistent to be of great practical value. They did, however, demonstrate the fact that the hard leguminous seeds show an increase in the percentage and the rate of gemination after treatment with concentrated H2S04 for varying lengths of time, and that in spite of the fact that there was great individual variation in regard to hardness, there is a certain maximum period of treatment with which damage results to the seed. A period of treatement ex- ceeding two hours proved dangerous in most cases. *. . 7 Work of a somewhat similar nature was done by Bergtheil and Bay in an article, "On the Cause of Hardness in the Seeds of Indigofera aurrecta in Annals of Botany? Vol. 21, p 57, they report the results of their investigations. They appear to credit the original use of H2S04 as a scarifying agent to hasten the germination of seeds to the studies of Dr. Butler on treating cotton seeds for the destruction of insect eggs. They found that after only a 20 minute to 30 minute treatment with concentrated H2S04, and consequent washing and drying, the germination is perfect. They point out also that the reasons for such an increased germination after treatment wi tli H2S04 were alleged by Percival to be due to the fact that the testae contain a relatively large ash constituent and hence resist the penetration of water until they are scarified by the action of the acid# This explanation, however, according to Bergtheil and D a y is not sufficient in the case under their observation, viz. that of Indigofera aurrecta. They proceed in turn to attempt an explanation of the impermeability by studying the structur e or organic constituents of the 3eed coat. By the use of stain- ing material it was decided that the impermeable layer is on the extreme outside of the seed coat, and the conclusion ar- rived at was "that the hard nature of the seeds of Indigofera aurrecta is due to their possession of a very thin outer cover- ing of a substance resistant to water-pro bably a transition product between cellulose abd cuticle. Scarifying removes a portion of this resistant covering and so allows penetration . 8 of water, while H2S04 does the same by causing rupturing and swelling or by converting it into a body akin to cellulose and hence permeable to water. Then, directly in keeping with these findings, Paine and Saunders, while experimenting with sources of material which would give a non-extensible membrane for a Pfeffer’s cell, found what they considered to be the solution for the problem. In an article, "On a Peculiarity Exhibited by the Testa of Wrinkled Peas" in Annals of Botany, Vol. 32, p 175, they maintain that the differences in permeability exhibited by the seeds coats of peas is due to a layer of waxy bloom on the surface of the seed. This conclusion was arrived at after it had been discovered that there was a marked differ- ence in the permeability of the seed coats of smooth and wrinkled peas. With smooth peas a uniform peimeability was obtained, whereas with wrinkled ones a very irregular and much netted permeability was evidenced. Upon further inves- tigation it was found that the depressions in the coats of wrinkled peas were impermeable while the ridges were permeable to a UaCl solution. Upon soaking in warm alcohol for several hours the permeability was perfectly uniform. It was decided that in the case of the smooth pea the bloom layer was readily rubbed off, whereas, with a wrinkled pea the wax would be rubbed only from the ridges, thus rendering thorn permeable. How as a result of this vast amount of work on permeabil- ity and the action of toxic substances in general, only frag- ments of which are cited above, there have arisen several more , * , . . * 9 or less definite problems* There is at present somewhat of a dispute as to the real cause of the impermeability of the seed coat of peas* One group holds that it is due to an external layer of some foreign impervious substance while a second group maintains that the property of impermeability is resident in the deeper layers of the seed coat itself and is not to be isolated therefrom. It is the purpose of the following study to find some conclusive evidences upon either of the above contentions. * . 10 II Citation of References. Arcichovskij . , V. - Biochemi sche Wirkung Hociist Konzentrierter Lb sungen. Biochem. Zeitscher, Vol. 50, 233. Bergtheil, C. and Day, D. L. - On the C a use of ’Hardness’ in the Seeds of Indigofera aur recta. Annals of Botany, Vol. 21, 57. u Bokorny, Th. - Uber den Einflusz verschiedener Substanzen auf die Keinung der Pflanzen3amen. Wachstumsforderung durch Einige. Biochem. Zeitschr. , Vol. 50, 1. Brown, A. J. - Semipermeable membrane in Graminae. Annals of Botany, Vol. 21, 79 Kahlenberg, L. and T r ue, R. H. - On the Toxic Action of Dis- solved Salts and their Electrolytic Dissociation. Bot- anical Gazette, Vol. 22, 81. Kurzwelly, W. - Uber die Wiederstandsfahigkeit trockener pflan- zlicher Organismen gegen giftige Stoffe. Jahrb. f. wiss. Bot. 1913. Vol. 38, 291. Love, H. H. and Leighty, C. E. - Cornell University Bulletin 312. Germanation of Seed as affected by sulfuric Acid Treatment. Paine, S. G. and S a unders, L. M. - Annals of Botany, Vol. 32, 175. On a peculiarity Exhibited by the Testa of Wrinkled Peas. * G f 11 III Methods and Materials. Since the purpose here is to demonstrate varying amounts, of permeability of the seed coat, it is convenient to use some agent which will definitely show its effects upon the seed. Such an agent is found in Sulfuric acid, and it was decided to use it in varying concentrations ranging from n/32 to cone, sp gr 1.84. The solutions which were used were i\/32, n/16, n/8, n/4, n/2, n/l, 2n, 4n, 8n, 16n, 24n, 32n, and concentrated or 37.5 noimal solution. Four varieties of pea seeds were used throughout the ex- periment. These were Vaughan’s Gradus, Alaska, Canada Held, and Black Bye Marrowfat. Each variety was treated for one and two hour periods by immersion in each of the above concentra- tions. The acid solutions as well as the geminating seeds were kept at a constant temperature in the constant tempera- ture cases. Two complete series wer run off - one at 20 ’c and one at 15’ c. The seeds were all rinsed in running tap water for a period of approximately five minutes following the aci<£ treat- ment. They were then placed on the regulation Rag-doll Seed Corn Tester for germination. The doll which was used, however, was the modified form consisting of the cloth strip wrapped with a strip of glazed wrapping paper upon a core of wire scree/ning. Such an arrangement prevents the spread of disease from one portion of the doll to another, as the paper is rolled up with the cloth and thus comes between each successive layer i ' t I I 12 of the doll. The seeds on the doll were thoroughly wetted he- fore wrapping, and were wetted also upon every alternate day during the germination period. Throughout the period the dolls v/ere kept in earthenware jars in the constant temperature cases. During the period of germination the 3ecds were examined every alternate day to determine the number which had geminated during each period. It was found that six days were sufficient to geminate all viable seeds of all varieties at both temper- atures. After six days in the dolls the seeds were removed, the dolls sterilized by boiling, and a new lot put into the gerrainatiors. Lots of 50 peas of each variety were used in all tests and at each observation of the number of germintated seeds was counted. As a criterion of gemination it was decided to use the rupture of the seed coat and a consequent elongation of the plumule to the extent of 2 ram. Seeds which showed such an elongation but which appeared to be no longer alive were not counted as geminated. The amount of gemination which is given in all cases is thus a positive one indicating subsequent growth of the seedling* Some work was also done with alcohol to find out whether the permeability was due to a bloom coat on the outside of the seed, Dor this purpose samples of the four varieties of seeds used above were immersed for two hours in absolute alcohol at an approximate temperature of 35’ C. The Seeds were washed in running tap water, dried, and immersed in concentrated H2S04 for one and two hours, and then geminated as above. . . • . . . * . To test and compare the results of acid treatment, check lots were run for both temperatures and for both periods of treatment. The seeds were soaked in distilled water for one and two hours and then treated exactly like the acid treated seeds# It will be noticed that in all cases it is assumed that the rate and per cent of germination, and the appearance and subsequent growth of the seedlings are direct indications of the extent to which the seed coat is permeable in each case# If the germination is low it is because the seed coat was readily permeable to the acid and vice versa. Account was also taken in each case of the comparative growth of mold on the seeds in order to determine if there is any relation be- tween the amount of this mold growth and the acid treatment* . - 14 IV Results and Experimental Data. The numbers here expressed indicate percentages of germ- ination in each case. They are given for periods of two, four, and six days following acid or water treatment .The temperature and the variety of seed are indicated at the top while the con entration of the solution which was used in each case is ex- pressed at the side. The length of time of treatment is also included at the top and is indicated as one hour or two hours. 15 Temperature 20 ’ C . Gradus Alaska 1 hour 2 hours 1 hour 2 hours 2 4 6 2 4 6 2 4 6 2 4 6 cone . to 94 98 6 92 94 72 82 82 48 84 84 32 n. 44 92 94 4 86 90 84 100 100 50 94 94 24 n. 32 94 94 14 90 92 60 90 96 68 94 94 16 n. 68 88 92 2 90 92 96 98 98 69 100 100 8 n. 80 92 92 16 90 90 96 98 98 70 96 96 4 n. 22 76 86 0 70 70 89 92 96 18 82 82 2 n. 30 76 76 0 53 53 80 86 86 24 62 62 1 n. 4 78 80 0 42 50 70 78 78 34 62 64 n/2. 20 64 64 0 36 42 62 42 84 26 54 62 n/4. 14 62 70 6 64 60 61 69 73 44 64 64 n/8. 16 94 94 12 64 64 84 96 100 42 82 84 n/16. 96 86 84 32 76 80 94 94 94 70 78 78 n/32. 80 86 86 58 78 80 92 94 94 76 80 84 check 14 92 92 8 82 82 80 98 98 60 94 96 check 2 84 90 62 84 88 24 92 92 98 98 98 16 Temperature 20’ C. Canada Field Black Eye Marrowfat 1 hour 2 hours 1 hour 2 hours 2 4 6 2 4 6 2 4 6 2 4 6 cone . 70 80 80 74 94 94 46 74 88 46 82 82 32 n. 82 96 96 72 88 88 71 91 96 48 84 84 24 n. 66 88 96 * 54 96 96 78 94 94 54 92 92 16 n. 90 96 96 38 92 94 82 88 88 30 92 92 8 n. 88 94 96 44 90 90 86 88 88 50 94 94 4 n. 74 92 92 14 76 78 48 56 • 56 4 46 46 2 n. 58 82 86 8 68 68 36 74 82 4 30 30 1 n. 44 74 74 16 50 54 18 50 52 0 12 14 n/2. 45 69 73 8 38 42 32 54 56 4 20 24 n/4. 40 60 68 34 64 68 42 54 60 22 32 36 n/8. 48 92 92 26 70 72 • 60 78 78 30 44 46 n/16. 88 98 98 46 78 82 80 82 82 50 56 58 n/32. 80 86 86 59 67 71 88 92 92 58 62 66 check 64 84 88 30 65 69 84 88 92 26 74 74 check 40 92 94 56 62 62 8 7S > 79 72 74 74 17 Temperature 15’ C. Gradus Alaska 1 hour 2 hours 1 hour 2 hours 2 4 6 2 4 6 2 4 6 2 4 6 cone . 54 94 94 38 90 90 78 84 84 38 90 90 32 n. 62 92 96 0 86 92 92 100 • 100 20 90 90 24 n. 0 76 92 4 88 90 14 98 98 22 90 90 16 n. 0 78 88 2 80 86 22 96 98 18 90 92 8 n. 22 90 90 0 60 92 58 96 96 4 96 ICO 4 n. 20 92 92 0 70 76 68 90 90 4 82 CO CO 2 n. 14 66 78 0 52 60 66 84 84 4 80 80 1 n. 10 76 78 0 42 52 70 86 88 0 66 68 n/2. 0 2 76 0 56 62 0 56 98 2 70 72 n/4. 0 0 74 0 24 54 0 42 94 0 64 72 n/8. 12 86 92 0 38 68 72 86 88 0 84 84 n/16. 20 82 90 0 30 54 80 94 98 0 86 90 check 12 80 84 12 78 78 64 98 98 66 90 92 18 Temperature 15* C. Canada Field Black Eye Marrowfat 1 hour 2 hours 1 hour 2 hours 2 4 6 2 4 6 2 4 6 2 4 6 cone . 90 90 90 79 100 100 80 84 86 48 90 90 32 n. 86 94 98 42 88 90 70 92 92 36 82 90 24 n. 20 94 96 26 90 90 6 98 100 30 87 87 16 n. 4 94 98 14 90 96 14 78 80 12 76 84 8 n. 58 86 92 4 78 94 52 92 96 0 84 88 4 n. 61 83 89 8 80 88 48 62 62 0 62 64 2 n. 54 84 90 2 57 67 32 72 72 0 32 34 1 n. 38 72 80 2 54 58 40 56 58 0 10 16 n/2. 0 24 70 2 50 56 0 12 56 0 28 35 n/4. 0 26 74 0 36 64 0 20 66 0 22 26 n/8. 40 82 86 0 32 52 52 76 80 0 36 42 n/16. 48 86 88 0 46 66 42 68 74 0 38 50 check 68 98 98 44 82 90 48 84 84 46 78 78 19 Results in percentages of germination after soaking for two hours in absolute alcohol at a temperature of about 35* C. and a subsequent treatment with concentrated Sulfuric Acid: 1 hour 2 hours 2 4 6 2 4 6 Gradus 0 68 80 0 72 74 Alaska 12 82 86 2 96 98 Canada P. 42 84 86 8 90 90 Black Eye M. 6 60 70 4 80 86 Mean cf final 20 * C.- Mean-4 . germinations for 1 hour. 15* C.- tests. Me an -2 a series ■1 hour, tests . Gradus 88 90 Alaska 98 97 Canada P. 96 82 Black Eye M. 80 82 20* C.- 2 hours. 15 1 C.- ■2 hours. Mean-3 tests . 1 test only. Gradus 86 78 Alaska 93 92 Canada F. 87 90 check tests: Black Eye M 82 78 20 V Discussion and Interpretation. The most apparent variation in germination occurring in these results is that one resulting from a change in the con- centration of the acid solution. Although the variation se- cued here is not nearly as striking as that reported by Arcic- hovskij, it is nevertheless a very real and constant variation. Thus in all cases, regardless of length of treatment and the variety of seeds which was used, the lowest percentage of germ- ination is secured by concentrations ranging from l/4 normal to 4 normal. Lower concentrations seem to have little effect upon the percent of germination while higher ones, especially up to a two hour soaking in concentrated acid, actually increase the number of germinated seeds above that secured in the check germ- inations. There is, of course, an occasional exception to these general statements, but these are very rare and possess no more than chance significance. A maximum germination is secured in nearly all cases by the use of concentrations ranging from 8 n. to concentrated acid. The seeds are killed, then, principally by the medium strength solutions, whereas the more highly con- centrated solutions actually enhance germination. Such a phenom- enon is due largely to the nature of the seed coat of the peas. This coat although not selectively permeable, and hence ex- ercising no semi-permeable activity in regard to the acid sol- utions, is nevertheless very impermeable to the acid. Especially is this the case with dry seed coats. When they once imbibe water from the surrounding solution the acid has a ready access to the interior of the seed, an d a lowering in the germination occurs. Such an explanation seems to fit in quite readily with the above results. A maximum amount of water is imbibed from concentrations . 21 below 8 normal, and the amount of acidin such solutions is suf- ficient to materially cut down the germination until a concen- tration of 1/4 normal is reached. Below this point the amount of acid is so small that there i s no apparent decrease in the germ- ination. In close relation to the variation of the percentage of ger- mination with a change in the concentration of the solution is the change in the rate of germination. The results show a very striking temperature effect in this connection. At 15’ C. the rate of germination is in nearly all cases materially slower than at 20* C. The rate of germination is also influenced by the length of the period of treatment. A two hour exoosure to acid usually results in a materially slower germination rate than that secured by a one hour treatment. Furthermore, the rate is slowest where the germination percentage is lowest. Such a slowing in the rate is usually manifested by a retarded beginning of the germination process . It is also apparent that the length of the time of treatment also has a very direct influence. Except in a very small number ol cases, there is a lower percentage of germination for the two hour treatment than for the one hour exposure. This is doubtless- ly due to the greater length of time during which the acid acts upon the seed coat in case of the longer periods of treatment, as well as the lengthened time for the imbibition of water in such a case. In check germinations there occurs no such differ, ence; in fact, the one huur soaking usually gives a higher per- centage of germination than the two hour treatment. In all cases, it will be noticed that the percent of germ- ination is higher for the treatment at 15 'C. than at 20*0. , 22 Although there is a similar variation in each temperature series, according to the strength of the solution, the percent, germin- ation for germination, is always higher for the 15’ series. This fact is due directly to the lessened chemical activity of t v e sol- utions at the lov/er temperature. Another very striking variation, and one to which few of the earlier investigators paid much heed, is the one occurring between the different varieties. This variation is quite marked and is constant throughout all concentrations and times of treatment, al- though it is not nearly as apparent with the higher and lower concentrations as it is in the dase of the most poisonous ones. With few exceptions the order of the percent of germination from highest to lowest is as follows: Alaska, Gradus, Canada Field, and Black Eye Marrowfat. Although the absolute percentages vary for different concentrations, times, and temperatures, their rel- ative rank always remains the same. Thus, although the lowest germination percent ranges from 1/4 normal to 2 normal, it always occurs in the casw of the Black Eye Marrowfat variety. This is an excellent demonstration of the varietal as well as individual variation of the seed coat characteristics. The growth of mold was also observed throughout the alcove experimentation. The greatest variation in this regard came in relation to the number of dead peas. Mold was not observed grow- inh on living peas in any case, and the amount hence always var- ied inversely with the number of germinated peas. Although there was a very profuse growth of mold in such cases at 20’, the am- ount of mold at 15’ was very slight even in the cases of minim- um germination. Bacterial contamination, as evidence by sliminess of the seed coat, was not materially decreased toy the lowering in tifimpprflt.nrfl. ‘ 23 Another very interesting occurrence is the process of pit- ting of the seed coat. This pitting occurs in the use of concen- trations ranging from 24 normal to concentrated acid, an d is most marked with the latter treatment. It is interesting to note that the amount of t v is pitting is quite closely correlated with the percentage of germination. The extent of the pitting seems to he directly indicative of the extent of acid injury. There is also a distinct individual difference in the amount of pitting. Some seeds are exceedingly pitted and almost charred by soaking in the concentrated acid, while others withstand even a two hour expos- ure with little apparent injury to the seed coat. Finally, there remains to be discussed the very interesting results of the work with alcohol. From the results, it will be observed that a treatment of two hours with absolute alcohol at a temperature of 35* C., followed by a subsequent soaking of one to two hours in concentrated acid, resulted in percentages of ger- mination not very far removed from those attained by the treat- ment with concentrated Sulfuric Acid without presoaking in alco- hol. The variation between the two is not much more than that whidh occurs between any duplicate series. Not only is the ger- mination percentage lowered in only a few cases, and then but slightly, but in an equal number of cases the percentage of ger- mination is actually increased after the alcohol treatment. It would seem on the basis of t ese results, that the con- clusion of Paine and Saunders in regard to the cause of the im- permeability of the seed coat of peas, does not hold for the var- ieties which were used in this work. Neitherwas it possible to establish any certain correlation between thd particular nature of the seed coat in each variety with its percentage of germin- * * 24 ation. Of the four varieties which were used, Gradus had the most wrinkled seed coat, while Alaska came second in thid regard. The coats of Can ada Field and Black Eye Marrowfat are smooth. There is no apparent relation between these facts and the results of the experiment-either in the case of check, straightacid, or alcohol plus acid treatment. It was furthermore impossible to establish any correlation between the amount of pitting, spoken of above, and the nature of the seed coat with reference to smoothness. It must therefore be concluded that the property which renders the seed coat of peas relatively impermeable to the entrance of acid, located within the structure of the seed coat, and is not an ex- ternal coat of wax, bloom, or other impervious material. 25 VI Conclusions. 1. The percent of germination is lowest for medium concen- trations, but this is due rather to an increased permeability of the seed coat than to ary intrinsic quality of greater poison- ousness on the part of such concentrations. 2. The percent of germination is higher with treatment at 15' than at 20' C., and the rate of germination is retarded by a decrease in the temperature. 3. An increase in the length of treatment with acid results in a lowering of the percent of germination. 4. There exists a marked variation in germination between the varieties of peas; the order from highest to lowest being: Alaska, Gradus, Canada Field, and Black Eye Marrowfat. Individ- ual variation is also very apparent. 5. Since presoaking for several hours with warm alcohol seem: to have no significant effect on the percent of germination, it is concluded that the impermeable property is not due to an ex- ternal coating of bloom or wax. 26 VII Figures. These figures are constructed from the data cited under the heading of Results and Experimental Data. They are in- tended to show in graphic form the respective curves of var- iation for the varieties of seed for different lengths of treatment in the consecutive concentrations of solutions. n o r Pe.v'C.e.n't of t* ^ O' >? O ft O 0 o o o 0 *J\Z tol & 2. rr} O ZD £v — S 1 1 1 1 s / g o ft -S’ n \ ' 'A \ ' M n°|_| I - 0-02 7 3>n?)!d znotf \ -'OoSJ V '5>no h x -'QoSl