XCbe xaniverstts of Chicago AFTER-RIPENING AND GERMINATION SEEDS OF A DISSERTATION SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL OF SCIENCE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BOTANY BY RIAL CATLIN ROSE Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from The Botanical Gazette, Vol. LXVII, No. 4 April 1919 Digitized by the Internet Archive in 2017 with funding from University of Illinois Urbana-Champaign Alternates https://archive.org/details/afterripeninggerOOrose BtL 3 Q. £ This paper gives the results of an attempt to determine the con- ditions favoring the after-ripening and germination of the seeds of Tilia americana , Sambucus canadensis , and Rubus Idaeus, and some of the chemical processes involved therein. Since layering of these seeds usually results in very low percentages of germination, it was thought possible to discover some other means of overcoming their dormancy. Literature The present state of our knowledge of the causes of delay in germination, and the means of overcoming it, is admirably sum- marized in a recent paper by Crocker (5) . He divides seeds which show delay in germination into 7 classes. In 3 of these the seed coats play the important role, while in the fourth dormancy is occasioned by the embryo. Where dormancy or poor germination is due to the seed coat, the use of concentrated sulphuric acid as a carbonizing agent has become a common practice. Rose (23) mentions Rostrup (24) as the first to resort to this treatment, and lists Todaro (25), Hiltner (12), Jarzymowski (14), Bolley (2), and Love and Leighty (19) as investigators applying the same method. Ewart (9) found this treatment effective with several 281 282 BOTANICAL GAZETTE [APRIL species of Acacia , as did Crocker (unpublished work) with Scirpus. The length of time required by this treatment varies from a few minues to several hours, depending upon the resistance of the coats. Boiling water or warm water, used as a forcing agent, has proved effective in a number of cases where hard-coatedness is the cause of the delay. Bruyning (3), working with the seeds of Ulex europaeus, found that a treatment of 1-5 seconds with boiling water raised the percentage of germination from 13 for untreated seeds to 53 • 5~7 5 • 5 f°r treated seeds. Honing (13) obtained his best results with Albizzia seeds by soaking them in water at 6o° C. for at least 3 hours, while with Mimosa 60-70° C. proved most effective, as did 70-75° C. for Pithecolobium . Soaking seeds of Crotalaria in warm water proved disadvantageous. Bolley (2) states that improvement in germination was obtained by this method if the exposure was not’long enough to kill the embryo. Nobbe (21) mentions Alexander von Humboldt as the first investigator to use chemicals as forcing agents. From the time of Humboldt (1793) up to 1873, the date of publication of Nobbe’s book, many investigators used as forcing agents a great variety of substances, both organic and inorganic. The range of substances used is more interesting than the results obtained. Moreover, quickly germinating seeds were used and in such cases the effect of forcing agents is not so striking as where dormancy is involved. Of the more recent workers in this field, Lehmann (16) was the first to emphasize the importance of chemical substances in connection with germination. He showed that the seeds of Ranunculus sceleratus are forced into germination by Knop’s solution, by soil, by soil wet with weak solutions of hydrochloric acid, potassium hydroxide, ferric chloride, and hydrogen peroxide. Two years later Gassner (10) found Knop’s solution effective on unthreshed seeds of Chloris ciliata , and more recently (11) has shown that for several other seeds various nitrogen compounds, especially nitrites and nitrates, are effective forcing agents. Chloris ciliata was found to have a membrane impermeable to potassium nitrate and magnesium nitrate, and from this Gassner concludes that the effect is upon the seed coat alone. Lehmann (17) and Lehmann and Ottenwalder (18), working with seeds representing iqiq] ROSE— AFTER-RIPENING AND GERMINATION 283 a number of different families, showed that acids in low con- centrations, especially hydrochloric acid, are effective forcing agents. Crocker and Davis (6) obtained similar results for seeds of Amaranthus (unpublished work) and Alisma. Bases are equally effective for Sagittaria and Alisma , but not for Amaranthus. According to Ottenwalder (22) bases exert an inhibitory effect on seeds of Epilohium hirsutum. In those cases where a state of dormancy exists in the embryo itself (< Crataegus and Malus ), temperatures slightly above freezing have been found effective in hastening after-ripening (7). In Crataegus , as Eckerson (8) has shown, the hypocotyl becomes more acid as after-ripening progresses; hence dilute acids hasten after-ripening by acting upon the hypocotyl directly. Material The seeds used in these experiments were gathered in the summer or the fall of 1916 and 1917. Each year those of Sambucus were all collected on the same day from neighboring plants. Tilia seeds of the 1916 crop were collected during October from trees growing on the dunes at the southern end of Lake Michigan. The 1917 crop was gathered during September from trees in the parks of Washington, D.C. The seeds of Rubus were collected during late June 1916 from neighboring plants of several varieties, but no attempt was made to keep those of the different varieties separate. Among the seeds of all 3 species were found many without embryos or with defective embryos. In most cases this fact accounts for the varying number of seeds used in the cultures. Approximately 60 per cent of Rubus , 75 per cent of the 1916 Sambucus, and 80 per cent of the 1916 crop of Tilia were viable. Not more than 5 per cent of the 1917 crop of Tilia and Sambucus were defective. Histology and microchemistry of seed coats Sambucus: Endocarp. — The seed in cross-section shows in the lignified endocarp 3 regions: (1) the outermost, consisting of 3 or 4 layers of cells of irregular size and shape, with thin walls and large lumina; (2) a middle one of 1 or 2 layers of fibers in cross-section; and (3) an inner one of 1 or 2 layers of fibers in longitudinal section. Seed coat. — This consists of several layers of collapsed cells with 284 BOTANICAL GAZETTE [APRIL lignified walls ; the cells contain a considerable quantity of reducing sugar. Tilia: Pericarp. — This is composed of two layers: (1) a surface region of loose fibers with cellulose walls, and (2) a thicker region of lignified fibers. Seed coat. — This consists of 3 regions: (1) cells with suberized or cutinized walls; (2) one layer of palisade cells with (a) outer end walls of cellulose, (b) a lignified light zone, (c) a pectinized region, and (d) a lignified region; and (3) 3 or 4 layers of cells with walls which stain with ruthenium red and give the ceric acid test. Rubus: Endocarp. — This consists of 2 layers: (1) an outer layer, variable in thickness, of lignified fibers longitudinally arranged in cross-section of the fruit; (2) an inner region of 4 or 5 layers of lignified fibers transversely arranged in cross-section of the fruit. Testa. — This consists of 4 regions: (1) 1 layer of cushion-shaped cells with lignified walls; (2) 4 layers of collapsed cells with cellulose walls; (3) 1 layer of collapsed cells with thick pectinized walls; and (4) 1 layer of cells with cellulose walls which appear as a thickened outer wall of the endosperm. Microchemistry In table I are given the results of the microchemical tests made upon the endosperm and embryo of each of the kinds of seeds used. Owing to the lack of a sufficient number of germinating seeds of Sambucus several of the tests have not been completed. The storage materials in all the seeds are very similar, starch, fats, and protein being found in every case. In addition to these Sambucus contains amylodextrin. Tilia contains much more fat and phytos- terol than either Sambucus or Rubus. The phytosterol shows up as a bright red layer around the fat globules when sections of the seeds are placed in concentrated sulphuric acid. Oxidase is present in the dry seeds in very small quantities, and in the germinating seeds benzidine gives a positive test only after several hours. Peroxidase, while present in dry Tilia seeds, is much more abundant in the germinating seeds. Dry seeds of Sambucus and Rubus give no peroxidase reaction. Catalase is found in both dry and ger- minating seeds of all 3 species. TABLE I ROSE— AFTER-RIPENING AND GERMINATION Rubus Germinated Embryo -t- _L "d + '+ ++++++++'! Endosperm + 1 + +H~+ + ^+++"j3 Ungerminated Embryo 1 1 1 +++++1 i+i Endosperm i i i +++++ , . +| Sambucus Germinated Embryo 1 ^ | 'v-^+‘G Endosperm s + i i ++++ i i i +i < Endosperm Alkaline Ungerminated Embryo + + + + + + + + + ~~ + + Neutral or acid Endosperm -4-4- ’cS -d + 1 1 +T++ l l ++S s + + fc O Substance Starch Amylodextrin Reducing sugar .... Protein reaction: Xanthoprotein . . . Biuret Fat Phytosterol Tannin Oxidase Peroxidase Catalase Reaction 286 BOTANICAL GAZETTE [APRIL Conclusive determinations in regard to the reaction of fresh dry- seeds of Tilia have not been made, but preliminary tests, where neutral red was used as an indicator, indicate that the endosperm and cotyledons are acid and the hypocotyl alkaline. Seeds kept in dry warm storage for 9 months show an acid reaction throughout. Hydrogen ion determinations, the data for which are given later, showed an acid reaction for the stored seed as well as for the germinating ones. As germination begins, the reaction of the embryo of Sambucus changes from alkaline to acid, but the endosperm remains alkaline. Both dry and germinating Rubus seeds are acid. A qualitative analysis of the ash of Tilia seeds showed iron, calcium, magnesium, potassium, and aluminium present. No tests were made for sodium. Experimental data Freshly harvested Tilia seeds with a moisture content of 10 per cent or less, or seeds kept in dry warm storage for several months, fail to germinate when placed on a moist substratum and kept at room temperature. This is true not only of seeds with coats intact, but for those with the coats chipped or entirely removed. Fungi and bacteria soon attack seeds with the coats broken and decay takes place in a few days. The percentage of water held by air-dry seeds is shown in table II. The seeds used for these deter- minations were dried in a partial vacuum at 8o° C. until the weight was constant. TABLE II Water content of air-dry Tilia seeds Condition of seeds Weight of air -dry seeds in gm. Water loss in gm. Percentage of water loss Coats off 1.2754* 1-8559 2 . I904 1.5024 0.0788 O. 1782 0.1574 O.II30 6.17 9 . 60 Coats on Coats on 7.18 Coats on 7-52 * Average of 4 duplicates. The variations in the percentage of water lost by the seeds with coats on is due to the presence of seed coats which contained no endosperm and embryo. That the failure of air-dry Tilia seeds, 1919] ROSE— AFTER-RIPENING AND GERMINATION 287 coats either on or off, to germinate is not due to an inability to absorb water is indicated by table III. The data given in this table were obtained by soaking seeds in distilled water at room tempera- ture until they had come to constant weight. Here again the TABLE III Water-holding capacity of air-dry Tilia seeds Condition of seeds Weight of air-dry seeds in gm. Water absorbed in gm. Percentage of water absorbed Coats off I . 2848* I . 2071 93-95 Coats on 2.1540 0.7841 36.40 Coats on I . 7842 0.4146 23.24 Coats on 2 .1198 O . 4804 22.66 Coats chipped I-4963 1 .5020 100.38 Coats chipped I . 5040 I-57I7 104.50 Coats chipped 1-9590 1.9185 97-93 * Average of 4 duplicates. variations in the percentage of water absorbed are in part due to the presence of seed coats which contain no endosperm and embryo. Even with the coats chipped it is not always possible to eliminate all empty coats or defective seeds. The fact that the coats interfere with water absorption to a considerable extent is clearly shown in the table. The fact that seeds with coats removed or chipped, however, and with a moisture content approximately equal to their air-dry weight will not germinate when placed on a moist sub- stratum at room temperature, is sufficient proof that water absorp- tion is not the only limiting factor to growth. That seeds that have been stored in the air-dry condition when the seed coats are intact can be forced to germinate is shown by the following experiment. Approximately 7000 seeds (200 gm.) of the 1916 crop, with pericarps removed and coats chipped, were placed on moist cotton in large Petri dishes and kept at 4-6° C. from March 24, 1917, to June 10, 1917, a total of 78 days. At the end of that time and before being transferred to a higher tempera- ture, several hundreds showed the hypocotyl protruding from the endosperm for 1.5-2. 5 cm. Of these, 100 were planted in soil in the greenhouse and 71 per cent produced seedlings. A second lot of 100 seeds was planted in soil out of doors, and 64 per cent 288 BOTANICAL GAZETTE [April produced seedlings. Two lots of 500 each were selected from the seeds in which the hypocotyl was still inclosed within the endosperm. These were planted in soil in the greenhouse and in the garden and gave 20 and 25 per cent germination respectively. All seeds not planted were again placed in cold storage. Twelve days later 400 with hypocotyls protruding from the endosperm were planted in soil in the greenhouse. Of these, 348, or 87 per cent, produced seed- lings within a week. By July 24, 1666 of these 700c cold storage seeds had germinated at a low temperature. Of the ungerminated seeds 100 placed on moist cotton at room temperature gave 31 per cent germination in one week. The roots of these were short 9 and thick and showed a great tendency to coil. At the same time air-dry seeds which had been stored at room temperature, when placed in soil or on moist cotton, decayed. Seeds kept in cold storage showed for the first few days a great tendency to mold, so that it was necessary to sterilize them with a 3 per cent solution of hydrogen peroxide for 1 hour on two separate occasions. With longer storage an immunity toward fungi is established, and although the coats may be covered with a thick layer of mycelia the endosperm and embryo are not attacked. Sections of the seeds examined under the microscope failed to show any hyphae present within the living tissue. On November 6, 1917, 6 cultures of 50 seeds each of both the 1916 and 1917 crops were placed in moist storage at 0-20 C., where they were allowed to remain for 140 days. At the end of that time no germination had taken place, which is in direct contrast with the result obtained in 1916 with seeds stored at 4-6° C. The failure to obtain germination here is interpreted as being due to the use of too low a temperature. The assumption that the exposure to this temperature was too long will hardly explain the results obtained, since if the temperature were not too low germination should begin as soon as the after-ripening process is complete. The results given in table IV, showing the percentage of germination obtained when these seeds were transferred to a temperature of 10-12° C., indicate that the storage temperature and not the length of exposure to it is the limiting factor. This conclusion is strengthened further by the following experi- ment. Unfortunately no count of the number of seeds germinated 1919] ROSE— AFTER-RIPENING AND GERMINATION 289 was made, as the experiment was used primarily for a different purpose. Approximately 1000 seeds of each of the 2 crops, stored under the same conditions as those indicated in table IV, showed no TABLE IV Seeds or Tilia stored at o-2°C. for 140 days; then at io-i2°C. Percentage of germination after Number of culture 1 2 days at 10-12° C. 19 days at 10-12° C. igi6 seeds 1917 seeds 1916 seeds 1917 seeds 1 66 24 74 28 2 68 20 72 24 3 70 12 80 18 4 74 34 82 34 5 70 26 76 30 6 19 22 56 30 germination after 140 days at a low temperature. When brought to the higher temperature the 1916 seeds germinated vigorously and in large numbers for the first 1 2 days and until the hypocotyls were 2-3 cm. long. From this point on no development took place and the seedlings gradually died. Here a temperature of 10-12° C. seems to be too low for continued growth. The 1917 seeds ger- minated much less vigorously, in fewer numbers, and only a few developed hypocotyls 2 cm. long. Comparing the results obtained in 1916 with those obtained in 1917, it is seen that the seeds after- ripen and germinate at temperatures slightly above freezing. Davis and Rose (7) working with Crataegus found that after- ripening takes place most rapidly at 3-6° C., and that temperatures considerably higher are more favorable for germination and growth. At 0-20 C. Tilia seeds after-ripen but do not germinate. At 4-6° C. after-ripening and germination both take place, the latter taking considerable time. After-ripened seeds germinate poorly at room temperature. Once germination has begun at the low temperature, growth is best at temperatures above 120 C. The germination of Tilia seeds depends, therefore, upon the proper regulation of the temperature, and can be accomplished by a period of after-ripening in moist storage at 0-20 C., followed by a sojourn of 2 or 3 weeks at 10-12° C. until germination is well under way, 290 BOTANICAL GAZETTE [APRIL and finally by a transfer to a still higher temperature in order to permit vigorous growth. These conclusions are drawn from the facts that (1) seeds after-ripened at 0-20 C. did not germinate until transferred to a temperature of 10-12° C.; (2) although germination began at the higher temperature, growth soon ceased; and (3) seeds which had been after-ripened and which had begun to ger- minate at 4-6° C. grew well when transferred to soil in the green- house. Table IV suggests that one-year old seeds are better than fresh, but additional data upon this point are desirable. A nursery- man with many years’ experience in the growing of trees and shrubs states that if Tilia seeds are allowed to become dry between the time of maturing and the time of layering a low percentage of germination results. On the other hand, if a high moisture content is maintained during this period no difficulty in germination is encountered. Up to the present time the author has been unable to obtain seeds which at the time of gathering had a moisture content of more than 10 per cent, and it seems probable that the water content of Tilia seeds is generally low at harvest time. While these seeds do not after-ripen to any considerable degree in air- dry storage, those that have been in the air-dry condition for a year after-ripen perfectly when put in a moist germinator at a low temperature. There seems to be no injury, therefore, even from protracted air-dry storage. No discussion is necessary to show that field conditions are not those most favorable for the obtaining of high percentages of germination. Neither does the nurseryman, when layering seeds, control the temperatures to the extent necessary to secure maximum results. Hydrogen ion concentration. — The determinations of the hydrogen ion concentrations were made with the hydrogen electrode. Twenty seeds were pulverized in a mortar, and, except in instances to be noted later, 25 cc. of water added. The temperature varied from 27 to 330 C., but in every case the necessary correction was made. The determinations were made upon the seeds in the unafter- ripened condition, after-ripened but not germinated, with hypocotyl 2 mm. to 5 mm. long, and with hypocotyl 0.5 cm. to 2 cm. long. Eckerson (8) has already shown that the acidity of the hypo- cotyl of Crataegus increases as after-ripening progresses. Her ROSE— AFTER-RIPENING AND GERMINATION 291 1919] determinations were made by the titration method with pheno- phthalein as an indicator. The PH of seeds with the hypocotyls 0.5 cm. to 2 cm. long is approximately 4 times as great as that of the unafter-ripened seeds. While this is not as great an increase as that found by Eck^rson, it may be due to the fact that her determinations were made upon the dormant organ alone, while here the whole seed was used, or to differences between the two kinds of seeds. Determinations made by the titration method would also probably give values much higher than those obtained by the hydrogen elctrode. Table V shows that the weight of the seeds increases as after- ripening progresses. This is not due to an increase in dry weight, TABLE V Concentration of hydrogen ion of Tilia seeds in different stages of after-ripening Condition of seeds 1 Air-dry 2 Air-dry 3 Air-dry 4 Air-dry 5 After-ripened 6 After-ripened 7 With hypocotyls 5 mm 8 With hypocotyls 5 mm 9 With hypocotyls 5 mm 10 With hypocotyls 0.5-2 cm 11 With hypocotyls 0.5-2 cm i2*With hypocotyls 0.5-2 cm 13* With hypocotyls 0.5-2 cm i4fWith hypocotyls 0.5-2 cm i5tWith hypocotyls 0.5-2 cm 16 After-ripened at room temperature (10 days) 1 7 After- ripened at room temperature (10 days) Weight in gm. pH 0.384 2 . 24X10-7 0-443 2 .OoXlO- 7 0-379 2.58X10-7 0-379 2.40X10—7 O.714 3. 24X10-7 O.752 3.02X10-7 0-943 6.76X10—7 O.932 5.75XIO-7 0 . 946 7.59X10-7 1-553 I.I8XIO-6 1.638 i . ioXio-6 I.578 9 33X10-7 1.709 9.33X10-7 1-450 1 .05X10—6 I-57I 1 .05X10— 6 0.689 2.51X10-7 0.642 2.51X10-7 1 *5occ. of water used. 1 100 cc. of water used; 25 cg. of water used for all others. since no photosynthesis has taken place, but to the large amount of water absorbed. Eckerson (8) likewise observed an increased water-holding capacity for the hypocotyl of Crataegus as after- ripening progressed. Of greater significance in this connection is the fact that, at least for the most advanced stage of after-ripening, variations in the amount of water used with the sample had little 292 BOTANICAL GAZETTE [APRIL effect upon the hydrogen ion concentration. With samples 10 and n, 25 cc. of water were used, with samples 12 and 13, 50 cc., and with samples 14 and 15, 100 cc. Although the variation of PH is considerable, it is by no means as great as that of the amount of water used, nor is it in the same direction. That the degree of dilution has no effect upon the PH suggests the presence of buffer salts, formed by the action of fatty acids produced during germina- tion and the constituents of the ash already mentioned. After-ripened seeds similar to those used in samples 5 and 6, which had failed to germinate when kept at room temperature for 10 days, gave a PH corresponding very closely to that shown by unafter-ripened seeds. This suggests that after-ripening is a reversible process, a fact to which Crocker (5) has called attention, and that a decrease in acidity may lead to secondary dormancy. Titratable acid. — Determinations of the titratable acid were made upon dry, after-ripened, and germinating seeds. For each determination the seeds were ground in a mortar with 10 cc. of water and titrated with N/10 sodium hydroxide with pheno- phthalein as an indicator. Titrations were made with freshly pre- pared samples and with others which had been allowed to stand for 48 hours. To the latter were added 10 drops of toluol and o. 5 cc. of N/10 hydrochloric acid. Table VI shows the number of cubic centimeters of N/10 sodium hydroxide necessary to neutralize the free acid in each sample. The figures are the average of duplicate determinations. Corrections have been made for the acid added. TABLE VI Condition of seeds Fresh samples After 48 hours Percentage of increase Dry O.41 0-45 1. 18 0.87 1.87 2.80 1 1 2 . 2 3I5-S 137.2 After-ripened Germinating While the amount of acid present is greatest in germinating seeds, it is seen that after autodigesting 48 hours the greatest percentage of increase over the freshly prepared samples is in seeds well after-ripened. Here is shown the fact that the after-ripened igig] ROSE— AFTER-RIPENING AND GERMINATION 293 seeds have a great power of increasing their alkali absorption, which may be due to lipase activity. Catalase. — Determinations of catalase activity of dry, after- ripened, and germinating seeds were made by means of Appleman’s apparatus (1). The samples, ground in a mortar, were all reduced to the same degree of fineness by rubbing them through bolting cloth. The catalase determinations were made at 250 C. To 5 cc. of water containing 0.02 gm. of pulverized seed material was added 5 cc. of Oakland dioxygen and the amount of oxygen released was measured after 1, 2, 3, and 5 minutes of activity. Appleman has pointed out that small amounts of acid greatly reduce or entirely destroy catalase activity. In order to remove this possible source of error the Oakland dioxygen used was neutralized by the addition of N/10 NaOH, or an excess of CaC03 was added to the meal. The data given in table VII are the averages of duplicate determinations. They show that dry, after-ripened, and germinating seeds, in the order named, exhibit increasing catalase activity. Eckerson (8), employing microchemical methods, arrived at similar conclusions for seeds of Crataegus. TABLE VII Condition of seeds Reaction of REAGENT Oxygen in cc. liberated AFTER 1 minute 2 minutes 3 minutes 5 minutes 1. Dry seeds Neutralized* 2-5 4.2 5-25 7.0 2. After-ripened (dried 2 days) . . . Neutralized* 7-1 ii .8 IS-4 21-5 3. After- ripened (dried 2 days) . . . With CaC03 6.8 11. 9 14.8 21.05 4. After-ripened (not dried) With CaC03 6-75 11 -3 I5-°5 20.75 5. Germinating With CaC03 19.4 27.86 31-5 37 03 *0.80 cc. N/xo NaOH to neutralize 25 cc. dioxygen. Drying after-ripened seeds for 2 days at room temperature has no effect on the amount of oxygen liberated, as is shown by com- parison of samples 3 and 4. Further evidence for the effect of the acid of the dioxygen upon catalase activity is shown in table VIII. Determinations made with after-ripened seeds not dried and with germinating seeds gave similar results. A comparison of the last 2 determinations show BOTANICAL GAZETTE [APRIL 294 that the neutralization of dioxygen or the addition of CaC03 is sufficient to eliminate any error due to the acidity of the reagent or the meal. TABLE VIII Effect of reaction of solution upon amount of oxygen liberated FROM DIOXYGEN BY Tilia SEEDS Condition of seeds Reaction of REAGENT Oxygen in cc. liberated after 1 minute 2 minutes 3 minutes S minutes After-ripened (dried 2 days) .... After-ripened (dried 2 days) .... After-ripened (dried 2 days) .... Not neutralized Neutralized With CaC03 2 . 1 7-1 6.8 3-i 11. 8 11. 9 3-6 15-4 14.8 4-3 21-5 21.05 .Oxidase activity. — The determinations of oxidase activity were made on dry, after-ripened, and germinating seeds in Bunzell’s (4) simplified apparatus with pyrogallol as the reagent. The material used, except in the case of the dried seeds, had been after-ripened at 0-20 C. for 140 days and then kept at 10-12° C. until a large percentage of the seeds had begun tQ germinate. After being dried in a vacuum over lime for 3 days at room tempera- ture the seeds were ground in a mortar and the determinations made on 0.02 gm. of meal. Table IX shows the readings in centi- meters of mercury after 3 hours and after 20.5 hours. TABLE IX Oxidase activity of dry, after-ripened, and germinating seeds of Tilia Time Dry seeds After- ripened Hypocotyls 1—5 mm. long Hypocotyls 0.5-2 cm. long After 3 hours O.52 1.03 2.01 i-39 After 3 hours o-53 I . IO I .92 i-52 After 20 . 5 hours 0.67 1-52 2-57 2.42 After 20.5 hours 0.68 i .t>7 2.52 2.07 During the experiment the temperature averaged 31.30 C. with a variation of =*=0.1 of a degree. Variations in the volume of air in the tubes due to this slight variation in temperature have been corrected by means of check tubes containing water only. The igig] ROSE— AFTER-RIPENING AND GERMINATION 295 results show that the oxidase activity rises with after-ripening and germination. Once germination has begun, no increase is to be noted. Discussion. — The results obtained show that the dormancy exhibited by the seeds of Tilia is not due to any property of the seed coat, although that structure may serve to lengthen the dormant period, but is to be ascribed to conditions obtaining within the endosperm or the embryo or both. In this respect Tilia resembles Crataegus, and the conditions necessary for after-ripening and germinating of the former are very similar to those required by the latter. Even with these conditions well known and various dif- ferences between dormant and after-ripened seeds clearly shown, it is still impossible to define the term after- ripening in anything more than general terms. The similarity of Tilia and Crataegus, with respect to the conditions necessary for after-ripening, does not permit one to conclude that the process in the two is the same. In any case after-ripening is not to be attributed to a change in any one condition, but to a series of changes which may vary for each individual case. Dormancy is to be looked upon, perhaps, as a condition of equilibrium in a series of chemical reactions; after- ripening as a displacement of this condition. Why low tempera- tures are effective in causing these changes and why the range of effective temperatures is so narrow are questions still to be answered. Sambucus Kinzel (15) states that for Sambucus nigra freezing for 2 winters is sufficient to bring only 39 per cent of the seeds to germina- tion. Even longer freezing is necessary for the seeds of S. racemosus. Results obtained by the writer in experiments to be described are very similar to those given by Kinzel, and show that in neither case have the conditions necessary for germination been even approximately determined. Nurserymen claim that layering results in almost perfect ger- mination if the seeds are not allowed to become dry between the time of maturing and the time of layering. Air-dry seeds are considered worthless. These statements are in a large measure 2q6 BOTANICAL GAZETTE [APRIL confirmed by the following experiments, although sufficient data are not yet available to warrant a final statement. Seeds removed from berries and allowed to dry at room tempera- ture for 2 days failed to germinate within 2 weeks when placed on moist cotton, although they never contained less than 22 per cent of moisture. Fresh seeds on moist cotton kept at 4-6°, 0-20, or 8° C. have never given more than 20 per cent germination when placed at room temperature or above. Air-dry seeds have given no better results. Although these seeds were kept at the low temperature for not less than 2 months, a longer period may be necessary. The experiments show that failure to germinate is not entirely due to injury resulting from drying, although that may be one of the determining factors. Neither is it to be attributed to inability of air-dry seeds to absorb water, since the quantity taken up in 48 hours by seeds with coats intact is equal to 38.55 per cent of their air-dry weight, while seeds with coats punctured absorb 39.16 per cent. Air-dry seeds contain approximately 6 per cent of water. The effect of layering is shown by the following experiments, in which the number of seeds used for the 1916 crop was 1000 and for the 1917 crop 5000. Two lots of air-dry seeds of the 1916 crop were mixed with soil. One lot was kept at 15-20° C., the other out of doors over winter. In spring the percentages of germination were 8 and 44 respectively. Fresh seed of the 1917 crop, which had not been permitted to become dry when treated in the same way, gave 51 per cent and 77 per cent respectively. Air-dry seeds of the 1916 crop one year old failed to show any germination. Loss of water seems to be accompanied by a reduction in vitality. Air-dry seeds gathered on October 14, 1916, were treated within 30 days with weak solutions of a large number of acids, bases, and salts. The acids used were malic, citric, tartaric, acetic, and butyric; the bases, potassium hydroxide, ammonium hydroxide, and sodium hydroxide; and the salts, sodium sulphate, nickel sulphate, ammonium sulphate, zinc sulphate, potassium sulphate, potassium nitrate, sodium nitrate, cobalt nitrate, ammonium nitrate, calcium chloride, sodium chloride, barium chloride, and potassium thiocyanate. The dilutions of the acids were N/ 200 ROSE— AFTER-RIPENING AND GERMINATION 297 1919] andN/400; of the bases, N/1000, N/2500, N/5000, and N/i 0,000; and of the salts, N/20 and N/200. The number of perfect seeds in the cultures varied from 43 to 96. In only 4 cases was the number below 60, and the average was 75. This variation is due to the presence of empty seed coats which could not be distinguished from the perfect seeds until they had taken up a considerable quantity of water. It was later found possible to candle the seeds and thus eliminate the majority of the empty coats. The candling was done by means of an incandescent light supported below a glass plate upon which the seeds were placed. Between the light and the plate was placed a vessel of water to prevent undue heating. The seeds were placed in 20 cc. test tubes containing the solutions and allowed to soak for 24 hours. At the end of that time the solutions were drawn off and the seeds distributed over the moist walls of the test tubes, which were then plugged with cotton and kept at a tempera- ture varying from 4 to 230 C. As soon as the seeds began to show signs of germination, they were removed from the tubes and placed in Petri dishes on moist cotton and kept at room temperature. Germination was slow, in the majority of cases extending over a period of 3 months. In the case of acetic acid, N/400, 58 per cent of the seeds germinated at the end of 176 days. The acids other than acetic showed little effect. The length of time over which bases can have any effect must be short, since in dilute solutions they are soon neutralized by the carbon dioxide of the air and that produced by the seeds. The cultures which showed germinations equal to or better than the checks are listed in table X. In order to test the effect of constant low temperature upon seeds soaked in solution of various chemicals, a second set of cultures was prepared in the manner already described and kept at 4-6° C. for 63 days. At the end of that time the tubes were placed at room temperature. To the list of substances used in the pre- ceding experiment were added potassium citrate, potassium tar- trate, potassium acetate, potassium chlorate, ammonium nitrate, potassium iodide, lithium chloride, ammonium chloride, magnesium chloride, sodium nitrite, and dipotassium phosphate, and also hydrochloric acid and sulphuric acid. The concentrations of the mineral acids were N/1000, N/2500, N/5000, N/ 10,000, and of the 298 BOTANICAL GAZETTE [APRIL salts N/20, N/200, and N/1000. Germination began 4 days after the cultures were placed at room temperature and continued for 18 days. At the end of that time in practically all of the cultures, in addition to the seeds which had germinated, others were found TABLE X Sambucus seeds in dilutions or acids, bases, and salts; TEMPERATURE 4-230 C. Substance Normality of solution Number of seeds Percentage of germina- tion Distilled water 78 12 Distilled water Sk IO Distilled water v-/o 68 IO Distilled water 13 Acetic acid N/ 200 79 0 18 Acetic acid N/400 72 28 Malic acid N/400 75 15 NH4OH N/ 2500 77 18 NaOH N/1000 88 19 NaOH N/2500 70 17 (NH4)2S04 N/20 75 28 (NH4)2SO, N/200 67 15 ZnS04 N/20 50 22 KNO, N/200 80 30 NaNO, N/20 46 19 NaN03 N/200 72 30 C0NO3 N/200 59 71 KCNS N/200 66 31 with the seed coat ruptured, but showing no sign of growth. All cultures in which a forcing effect of the solution is indicated by the germination of 20 per cent or more of the seeds are listed in table XI. Out of 13 other substances not given in the table, 5 showed results equal to or better than the average of the checks in at least one dilution. The nitrates and sulphates are again found among the more effective substances. So far as the nitrogen compounds are concerned, these results agree with those of Gassner (10) for seeds of Chloris ciliata. Potassium nitrate, mercuric chloride, and potassium iodide used in connection with alternating temperatures had even less forcing effect than the substances given in table XI. The concentrations used were for potassium nitrate N/20, N/100, N/200, N/500, 1919] ROSE— AFTER-RIPENING AND GERMINATION 299 N/1000, N/2000; for mercuric chloride N/400, N/1000, N/2000, N/4000, N/10,000; and for potassium iodide N/20, N/100, N/500, N/1000, N/2000. Three sets of cultures were set up in duplicate. TABLE XI Sambucus seeds in acids, bases, and salts , Substance Normality of solution Number of seeds Percentage of germination Percentage of seeds with ruptured coats Total percentage of seeds affected HC1 N/5000 6S 17 6 23 H2S04 N/2500 66 18 IO 28 H2S04 N/ 10,000 63 15 6 21 nh4oh N/ 1000 89 23 16 39 nh4oh N/5000 96 13 8 21 C6H809 N/400 IOI 21 4 25 kno3 N/20 78 28 ‘ 25 53 KNO, N/ 200 77 13 18 3i KN03 N/ 1000 86 24 8 32 C0NO3 N/200 92 4 44 48 C0NO3 N/ 1000 87 24 23 47 nh4no3 N/200 108 14 39 53 nh4no3 N/1000 56 14 12 26 NaN03 N/20 96 20 18 38 NaN03 N/200 83 33 20 53 NaN02 N/200 72 25 26 5i NaN02 N/ 1000 65 9 18 27 Na2S04 N/ 200 94 10 10 20 NiS04 N/20 84 10 10 20 NiS04 N/200 86 7 34 41 NiS04 N/1000 76 15 10 25 !\II,l-SO, N/200 85 5 37 42 ZnS04 N/20 86 41 1 42 KCL N/20 80 0 22 22 LiCl N/1000 80 0 22 22 NaCl N/200 89 1 37 38 NH4C1 N/20 97 2 24 26 NH4C1 N/200 84 13 15 28 Potassium citrate N/20 84 0 21 21 Potassium citrate N / 1000 95 7 37 44 KC103 N/1000 81 9 18 27 KI N/20 89 10 33 43 KCNS N/200 90 9 12 21 K2HP04 N/20 83 1 24 25 K2HP04 N/ 1000 55 0 34 34 Distilled water 70 0 7 7 Distilled water 85 8 7 1 7 Distilled water. . 109 1 14 x I 15 One set was kept at 20° C. and a second at 30° C. The third set was kept at 20° C. for 18 hours and at 30° C. for 6 out of every 24 hours. The air in the tubes was changed every second day. The 3°° BOTANICAL GAZETTE [apri duration of the experiment was 38 days. At the end of that time the only germinations obtained were those in the potassium nitrate, and in no case did these exceed 4 per cent. The seeds in the stronger mercuric chloride solutions were killed. The role played by the coat in the behavior of the seeds has not been determined. Of naked embryos placed on moist cotton 32 per cent developed chlorophyll within a week, formed the hypocotyl arch, and attained a length of 5-10 mm. Naked embryos pre- viously soaked in dilutions of hydrochloric acid and butyric acid and then placed on moist cotton gave no better results. Seeds treated with concentrated sulphuric acid for 4-60 min- utes and then kept under various conditions in regard to light, temperature, and oxygen pressure have never given over 20 per cent germination. A slight forcing effect by low concentrations of sulphuric acid was observed on seeds previously treated with con- centrated sulphuric acid for 2-14 minutes and kept in the light at room temperature. Seeds immersed for 5 minutes in water at 40° C. in 55 days gave 25 per cent germination. Reheated at the same temperature for 3 minutes, 33 per cent germinated after 40 days. Longer heating at 40° C. or up to 70° C. gave lower per- centages of germination. Untreated seeds gave no germination in the same length of time. These results emphasize the following facts concerning the conditions necessary for the germination of Sambucus seeds: (1) air-dry seeds with a moisture content of 6 per cent or fresh seeds with a moisture content of 22 per cent will not germinate when placed on a moist substratum at room temperature; (2) this is not due entirely to injury resulting from drying, although that may be one of the determining factors ; (3) air-dry seeds are able to absorb water to the extent of approximately 40 per cent of their air-dry weight, indicating that failure to germinate is not due to lack of water; (4) the effect of chemicals upon air-dry seeds is not marked, a slight forcing effect of several acids, bases, and salts has been observed, among which substances are found nitrates and sulphates ; (5) the role played by the coat in the behavior of the seed has not been fully determined; (6) a slight forcing effect by low concen- trations of sulphuric acid and by water at 40° C. has been observed; iqiq] ROSE— AFTER-RIPENING AND GERMINATION 301 (7) seeds remaining in contact with moist soil out of doors over winter gave 77 per cent of germination the next spring; whether this result is due to the low temperature, to certain constituents of the soil, or to a combination of these or other factors one cannot say. The results obtained by Kinzel (15), together with those just summarized, show that as yet the conditions necessary for the germination of Sambucus seeds are not fully determined. To permit the water content of the seeds to fall below an undetermined critical point may lessen their viability. However, that some other condition or combination of conditions is responsible for the low percentages of germination must not be overlooked. Kinzel’s suggestion that prolonged freezing is necessary should be given due consideration. Rubus Seed fruits of Rubus Idaeus, like the seeds of the 2 species already discussed, fail to germinate when placed on a moist substratum. It was determined that this is not due to an immature condition of the embryo. If the pericarp is left intact all treatments with low concentrations of acids, bases, and salts, immersion in warm water, cold storage, exposure to increased oxygen pressure, or to ether vapor, freezing and thawing, and injection with water under pressure are ineffective. When buried in the soil at 15-20° C. or out of doors over winter, a low percentage of germination takes place if the seeds are kept moist. Two lots of 720 viable seeds buried for 140 days under these conditions gave respectively 40 per cent and 20 per cent germination. Of 2 similar lots of seeds buried in tightly stoppered bottles, one at constant, the other at varying temperatures, none germinated when planted in the soil in the greenhouse. That these results are not due to injury resulting from drying or to inability to absorb water is indicated in ta,ble XII. The removal of the endocarp was accomplished by soaking the seeds in con- centrated sulphuric acid for approximately 2 hours. Following this treatment the seeds were washed quickly in a large amount of running water to prevent heating, then immersed in a 5 per cent solution of sodium bicarbonate to neutralize the remaining acid, BOTANICAL GAZETTE [APRIL 302 and finally rinsed in running water for 15 or 20 minutes. The carbonized endocarp was removed by rubbing the treated seed on filter paper. The selection of perfect seeds was now an easy matter. Table XII shows that the water-absorbing power for the seeds with the endocarp removed is 36-37 per cent of their air-dry weight, while that for the seeds with the endocarp intact reaches TABLE XII Water content and water holding capacity of Rubus seeds Condition of seeds Weight of air-dry seeds in gm. Weight of seeds dried in vacuum at 750 C. Percentage of water in air-dried seeds Weight of soaked seeds in gm. Water absorbed by air-dry seeds in gm. Percentage of water absorbed by air-dry seeds Endocarp removed . 0.6686 0.5842 12.62* 0.9120 O.2434 36.40* Endocarp removed . 0.6864 O . 6009 12.45 O.9414 0.2550 37 15 Endocarp intact. . . . I . 0464 0.9328 IO.85 I-5053 0.4589 43-85 Endocarp intact. . . . 2 . 0960 1.8680 IO.87 3 . 0080 O.9120 43-51 * On basis of air-dry weight. almost 44 per cent of their air-dry weight. From this it follows that the water absorbing power of the endocarp is greater than that of the seed with the endocarp removed. There is no evidence to show that the endocarp possesses any structure which would prevent the water absorbed by it from being passed on to the seed. Although seeds with the endocarp intact will not germinate, when that structure is removed by means of the sulphuric acid treat- ment germination takes place within a few days, as is shown in table XIII. The greater amount of the germination takes place between the fourth and tenth days. In seeds germinating after the tenth or twelfth day, growth is usually slow and the seedlings are weak. Failure to secure 100 per cent germination is due to the fact that during the removal of the carbonized endocarp in almost every case the seed coat is ruptured and the endosperm exposed to the attacks of bacteria and fungi. With the carbonized endocarp intact, uncertainty as to the extent to which the acid had penetrated and the inability to determine the number of fruits containing viable embryos lead to greater error than that occasioned by the attacks of the bacteria and fungi. igig] ROSE— AFTER-RIPENING AND GERMINATION 303 Muller (20) has recently pointed out that in various seeds that germinate readily the outward pressure of the contents at the time of rupture was but slightly greater than the breaking strength of the water-saturated coat, and Crocker and Davis (6) have found that seeds of Alisma are held in a dormant condition because the force of the expanding contents is not sufficient to rupture the coats. TABLE XIII Seeds of Rub us Idaeus with endocarp removed; ioo SEEDS PER CULTURE; TEMPERATURE 18-23° C. Treated with acid Percentage of germination after 4 days 6 days 8 days 10 days 20 days May 4 70 84 96 Mav 13 2 45 52 63 70 May 13 32 48 53 6l 61 May 13 24 46 55 55 May 24 50 73 83 88 May 24 20 63 77 84 May 24 ' . 22 61 80 May 24 40 78 86 88 May 24* 46 78 86 93 May 24* 57 77 82 89 Tune o 50 ! 86 95 j y * In darkness. Failure to absorb water is not the limiting factor, since both reach saturation after about 5 hours’ soaking. Two facts indicate that Rubus seeds belong in the same class with Alisma. In the first place they germinate readily once the endocarp is removed, and in the second place even with the endocarp intact they absorb water readily. Occasionally ungerminated seeds with the endocarp removed have been found which when examined closely show no break in the coat. This suggests that the inner pectinized layer of the coat may play a part in the delay, either by limiting water or oxygen absorption, or both. As already indicated, the removal of the carbonized endocarp resulted in the rupture of the coat in practically 100 per cent of the seeds. This renders extremely difficult the determination of the part played by that structure. Table XIV shows that the substrata most favorable for germina- tion of naked seed are cotton, filter paper, and quartz sand. An 304 BOTANICAL GAZETTE [APRIL inhibitory effect is shown by garden soil, clay, and greenhouse soil, the effect of the last named being greatest. These soils acidified gave no better results. Calcium carbonate used on filter paper or in sand to neutralize any acid present in the medium or remaining on the seeds after the sulphuric acid treatment had no inhibitory effect. Glass wool moistened with a boiling water extract or a cold water extract of greenhouse soil cut down the percentage of germi- nation to less than 50. Moreover, the seedlings were weak, with enlarged and discolored roots. In many cases germination started, but the roots were killed as soon as they came in contact with the substratum. Bone meal had been added to the greenhouse soil and this probably accounts for the injurious effect of the soil and the extracts. As is shown in table XIV, soaking in water for 24 hours TABLE XIV Effect of substratum upon germination of naked seeds of Rubus Idaeus; 106 SEEDS PER CULTURE; TEMPERATURE 18-23° C. Substratum Percentage of germination after 6 days 8 days 10 days 12 days 25 days Filter paper Filter paper with CaC03 Quartz sand Quartz sand Quartz sand with 5 per cent CaC03 . Greenhouse soil Greenhouse soil Greenhouse soil, acid Hot water extract greenhouse soil. . Cold water extract greenhouse soil . Garden soil Garden soil, acid Clay 50 3i 48 42 43 77 84 80 83 78 86 87 83 88 80 4i 48 90 90 85 89 29 10 95 92 92 93 88 25 1 1 43 48 29 10 19 previous to planting in garden soil raises the percentage of germina- tion to 55. On the other hand, soaked seeds planted on moist cotton gave 71 per cent as against 72 per cent for unsoaked seeds. Germination was at practically the same rate in the two cases. Seeds planted on 5 per cent agar gave almost as high percentages as those on 1 per cent. The results given in tables XIV and XV show that the water supply is not the limiting factor. ROSE— AFTER-RIPENING AND GERMINATION 305 1919] Seeds in the soil are more exposed to attacks by fungi than those on agar or cotton. Previous soaking shortens the time the seeds must lie in the soil before germination begins, and hence lessens the chance for infection. Unsoaked seeds placed on moist cotton TABLE XV Effect of water supply upon germination of seeds of Rubus Idaeus; IOO SEEDS PER CULTURE; TEMPERATURE 20-25° C. • Substratum 1 per cent agar* 1 per cent agar* 2 per cent agar* 2 per cent agar* 5 per cent agar* 5 per cent agar* Soaked 24 hours, then in garden soil. . . Soaked 24 hours, then on moist cotton Not soaked, on moist cotton * Average of 2 duplicate determinations. Percentage of germination after 6 days 8 days 1 0 days 1 2 days 16 days 37 49 70 70 70 22 50 62 62 30 37 50 50 50 40 64 73 73 35 42 60 60 32 52 61 61 12 36 47 50 55 43 64 70 7i 7i 32 66 72 72 absorb water easily, hence swell more rapidly than in the soil, and moreover are less liable to infection. Under these conditions soak- ing offers no advantage. Summary General. — Air-dry seeds of Tilia americana , Sambucus canaden- sis, and Rubus Idaeus do not germinate when placed on a moist substratum at room temperature. In no case does water absorption seem to be the limiting factor. Air-dry seeds planted in the soil over winter give low percentages of germination. Tilia. — Seed coats are not the cause of dormancy, although they may serve to lengthen the dormant period. A state of dormancy exists in the endosperm or embryo, or both. Seeds with coats removed after-ripen at temperatures slightly above freezing. At 0-20 C. seeds after-ripen, but do not germinate. At 4-6° C. both after-ripening and germination take place. Seeds after-ripened at 0-20 C. germinate readily at 10-12° C., but very poorly at room temperature. Once germination has begun growth proceeds best at temperatures above 120 C. 3°6 BOTANICAL GAZETTE [APRIL As after-ripening progresses the hydrogen ion concentration increases, as do also the water holding capacity and the oxidase and catalase activities. The greatest amount of free acid is present in the germinating seeds. Autodigestion of pulverized seeds shows the greatest acid increase in the after-ripened ungerminated seeds. This is probably due to their high lipase activity. Sambucus. — As high as 77 per cent of germination was obtained by layering fresh seeds out of doors over winter. No satisfactory forcing agent has yet been found. A slight forcing effect of several acids, bases, and salts has been observed. The best of these forcing agents are nitrates and sulphates. Although Sambucus seeds are probably injured by drying, that is not the only factor to be considered, since freshly gathered seeds with a moisture content of 22 per cent will not germinate when placed on a moist substratum. As yet it has been impossible to approximate perfect germina- tion, and much still remains to be learned concerning the conditions necessary to reach it. Rubus. — Dormancy is probably due to the high breaking strength of the endocarp. Seeds treated with concentrated sul- phuric acid for 2 hours, then thoroughly washed, germinate readily on cotton, filter paper, or quartz sand. The optimum temperature for germination lies between 20 0 and 2 50 C. Seeds germinate equally well in light or darkness. Naked seeds germinate poorly in soil. This may be due to the action of fungi, bacteria, or to other causes as yet unknown. As a practical method for the germination of Rubus seeds, if one is not to resort to layering, the writer suggests the following: The seeds should be removed from the pulp as completely as possible. If the berries are crushed and then thrown into water most of the pulp can be floated off. The pulp still clinging to the seeds may be removed by allowing fermentation in water to take place or by treating the seeds with a 5 per cent solution of sodium hydroxide for 15-20 minutes, after which they should be thoroughly washed in running water. It is essential to dry the seeds for at least 24 hours, or the treatment with concentrated sulphuric acid which follows 1919] ROSE— AFTER-RIPENING AND GERMINATION 307 will result in heating. The seeds should be left in the acid for approximately 2 hours. In order to obtain uniform results it is advisable to use a large excess of acid and to prevent the seeds from gathering in clumps or layers. Frequent stirring is essential. By rubbing a few of the seeds in the palm of the hand from time to time it is possible to determine when the entire endocarp on a majority of the seeds has been carbonized. When this point is reached the acid should be drained away and the seeds thrown into an excess of cold water. It is advisable to change the water frequently or to put the seeds in running water, where they should be left for at least 15 minutes. When they are removed from the water they should be treated with an excess of a 5 per cent solution of sodium bicarbonate until bubbles cease to rise, after which they may be washed in running water for 15 minutes. In order to remove the carbonized endocarp the seeds may be placed on filter paper and rubbed under the fingers. It is impossible to remove the endocarp if it has been allowed to become dry follow- ing the last washing. The writer is indebted to Dr. William Crocker and Dr. Sophia H. Eckerson for many helpful criticisms and suggestions during the progress of the work. Missouri State Fruit Experiment Station Mountain Grove, Mo. LITERATURE CITED 1. Appleman, C. 0., Some observations on catalase. Bot. Gaz. 50:182-192. 1910. 2. Bolley, H. L., The agricultural value of hard coats in alfalfa and clover seed. Paper read before the Association of Seed Analysts, 1910. 3. Bruyning, J. F. On the use of hot water for forcing germination in hard coated seeds. Jour. Landw. 41:86. 1896. 4. Bunzell H. H., A simplified and inexpensive oxidase apparatus. Jour. Biol. Chem. 17:409-411. 1914. 5. Crocker, W., Mechanics of dormancy. Amer. Jour. Bot. 3:99-120. 1916. 6. Crocker, W., and Davis, W. E., Delayed germination in the seeds of Alisma Plantago. Bot. Gaz. 58: 285-321. 1914. 7. Davis, W. E., and Rose, R. C., The effects of external conditions upon the after-ripening of the seeds of Crataegus mollis. Box. Gaz. 54:49-62. 1912. [APRIL 308 BOTANICAL GAZETTE 8. Eckerson, S. Ii., A physiological and chemical study of after-ripening. Bot. Gaz. 55:286-299. 1913. 9. Ewart, A. J., On the longevity of seeds. Victoria, Australia. 1908. 10. Gassner, G., Untersuch ungen iiber die Wirkungen des Lichtes und dcs Tempera turwechsels auf die Keimung von Chloris ciliata. Jahr. Hamb. Wiss. Anst. Beih. 3. 29:1-121. 1911. 11. , Einige neue Falle von Keimungsauslosender Wirkung des Stick- stoffverbindungen auf Lichtempfindlicher Samen. Ber. Deutsch. Bot. Gesells. 33: 217. 1917. 12. Hiltner, L., Die Keimungsverhaltniss der Leguminosen und ihre Be- einfliissung durch Organismenwirkung. Arbeiten. Biol. Abt. Land. Forstw. 3:30. 1902. 13. Honing, J. A., The warm water treatment of the seeds of certain herbaceous and green manure plants that are difficult to germinate. Meded. Deli- Proefstat. Medan. 10: 16-23. 1916; review E.S.R. 36:430. 1917. 14. Jarzymowski, A. von., Hartschalligkeit von Leguminosensamen und ihre Beiseitigung. Inaug. Diss. Halle. 1905. 15. Kinzel, W., Frost und Licht als beeinfliissende Krafte bei der Samen- keimung. Stuttgart. 1913. 16. Lehmann, E., Zur Keimungs physiologie und biologie von Ranunculus sceleratus und einigen anderen Samen. Ber. Deutsch. Bot. Gesells. 27: 476-494. 1909. 17. — — — -, Uber katalytische Lichtwirkung bei der Samenkeimung. Biochem. Zeitschr. 50:388-392. 1913. 18. Lehmann, E., und Ottenwalder, A., Uber katalytische Wirkung des Lichtes bei der Keimung lichtempfindlicher Samen. Zeitschr. Bot. 5:337-364- 1913- 19. Love, H. H., and Leighty, C. E., Germination of seeds as affected by sulphuric acid treatment. N.Y. (Cornell) Exp. Sta. Bull. 312. 293-336. 1912. 20. Muller, G., Beitrage zur Keimungsphysiologie. Untersuchungen iiber die Sprengung der Samen und Fruchthiillen bei Keimung. Jahrb. Wiss. 601.54:529-644. 1914. 21. Nobbe,* F., Handbuch der Samenkiinde. 22. Ottenwalder, A. von., Lichtintensitat und Substrat bei der Licht- keimung. Zeitschr. 601.6:785-848. 1914. 23. Rose, D. H., A study of delayed germination in economic seeds. Bot. Gaz. 59:425-444. 1915. 24. Rostrup, O., Rept. Danish seed control for 1896-1897. pp. 37. 1898; review E.S.R. 10: 53-54. 1898. 25. Todaro, F., Azione dell acido solforico concentrato su alcuni semi e in particulare sopra i semi duri dell Leguminosae. Staz. Sper. Agric. Ital. 34:613-689. 1901.