581.3 G17e JNIVERSITY OF ■__'NOi3 LIBRARY A i URBANA-CHAMPAIQN BOOKSTAGKS Digitized by the Internet Archive in 2013 http://archive.org/details/effectoflightongOOgard 3 Ube rantversttg of Chicago EFFECT OF LIGHT ON GERMINATION OF LIGHT-SENSITIVE SEEDS 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 WRIGHT AUSTIN GARDNER Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from The Botanical Gazette, Vol. LXXI, No. 4, April 1921 .«♦*.- *>*«»* Return this book on or before the „,. reasons for *"'P 1 ' B " h y Uni v.,sify. -" ,t '" <,iSm ' SSa, ! r nnr,o.s Library VI I NATION EEDS •.NCE L16l __O-l096 Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from The Botanical Gazette, Vol. LXXI, No. 4, April 192 1 v .v? v .- «&a» ZEbe 1Hntt>ersftE of Gbtcaao EFFECT OF LIGHT ON GERMINATION OF LIGHT-SENSITIVE SEEDS 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 WRIGHT AUSTIN GARDNER Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from The Botanical Gazette, Vol. LXXI, No. 4, April 1921 VOLUME LXXI NUMBER 4 THE Botanical Gazette APRIL 1Q21 effect of light on germination of light-sensitive seeds CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 279 Wright A. Gardner Historical Various explanations have been offered for the germination of light-sensitive seeds, and several conditions have been shown to favor or make possible the germination of such seeds. Rupture of coats, increased water supply, variation of quantity and intensity of light, reciprocal relation of heat and light, reaction of substratum and embryo, activation of enzymes, increased oxygen pressure, increased carbon dioxide pressure, and "certain inhibiting agencies" have been suggested as factors affecting the germination of light- sensitive seeds. Although quite possible, it seems hardly probable that no one of these is the fundamental or controlling factor. It would seem quite probable that one or two of these agencies are fundamental and the others are accessory means of setting in motion the processes that finally bring about germination. Enzyme action has been suggested repeatedly as a fundamental cause of germination, but no one has ventured to demonstrate the relation of enzymes to the germination of light-sensitive seeds. An attempt has been made in this investigation to discover the fundamental relation of light to the germination of seeds, and to show just what light does to start germination. The effect of light on the germination of seeds has interested botanists for many years. 249 250 BOTANICAL GAZETTE [april The first known publications on this subject were made by Casfary (4) in i860, when he announced that the seeds of Bulliarda aquatica are strongly light sensitive. In 1867 he (5) discussed the germina- tion of seeds of Pinguicula vulgaris. In 1876 Nobbe (38) made the statement that germination was neither favored nor influenced by light. After Wiesner (51) had published the statement that the germination of seeds of Viscutn album is favored by light, and Stebler (48) had shown that Poa pratensis and P. nemoralis germinated up to 60 per cent in light and only up to 7 per cent in darkness, Xobbe (39) published results of experiments with grass seeds, including Poa pratensis, Zea Mays, and some other large seeds to uphold his earlier contention. Pauchon's (41) results supported Nobbe in the controversy. In 1883 Cieslar (6) con- firmed and extended Stebler's results, reporting A grostis stolonifera and Nicotiana macro phylla as light sensitive. He made a careful study of the influence of temperature in connection with light, and showed that small seeds poor in reserve materials germinate better in white light, while large seeds are usually indifferent to light, and that seeds of Poa nemoralis germinate better in yellow light than in violet. Liebenberg (35) in 1894 confirmed Stebler's results, but referred to them as temperature effects. In 1893 Jonsson (23) showed that after-ripening has a definite influence on the action of light in germination, that light increases the percentage of germination, that heat rays are unimportant, that intermittent light is as effective as continuous light, and that intermittent temperature may be substituted for light in the germination of such seeds as Poa pratensis, P. nemoralis, A grostis stolonifera, and Daucus Carota. In 1899 Heixricher (19) began publishing the results of his work on light germination. He (20) reported that seeds of Pit- cairnia maydifolia germinate only in light, that the germination of Veronica peregrina seeds is hastened and several other small seeds are favored in germination by light. He considered the factors to be age, quickness of drying, moisture, illumination of parent plant, and light of different colors. He (21) concluded that the effect of light is a matter of activation of reserve materials, that the benefit of light is not due to its causing early carbon assimilation, i 9 2i] GARDNER— GERMINATION 251 but rather to its effect on the enzyme activity in the production and digestion of stored foods. Raciborski (43) found that tobacco seeds germinate in diffused light after 1-5 hours' illumination, a longer time being required if the intensity of light is low. In 1900 Tammes (49) declared that the exposure of dry seeds to direct sunlight did not affect their later germination, and in 1902 Laurent (29) made the same statement. Remer (44) reported that light hinders the germination of Phacelia tanacetifolia, but offered no explanation of the light relations. Laschke (28) confirmed earlier results with Poa, and stated that light cannot be replaced by higher temperatures. In the same year Figdor (10) made a report on the influence of light on the germination of seeds of Gesneriaceae. In 1912 he (11) reported that seeds of 12 species of this family are favored by light. In 1908 Bessey (3) found that seeds of the epiphytic Ficus aurea and F. populnea germinate only in light. Kinzel (24) in 1907 reported that the germination of freshly harvested seeds of Nigella sativa was prevented by light, while similar seed germinated up to 94 per cent in darkness. Even three minutes' illumination after 24 hours' incubation in darkness had a marked retarding effect. He considered the effect of light as photo- chemical, and designated such seeds as "light hard." The germina- tion of some light-sensitive seeds in darkness was hastened by soaking in a solution of an enzyme such as papayotin (25). He (26) also published a long list of light-sensitive seeds, to which still others were added in his later work (27). He recognized as important factors in germination of seeds age of seed, character of seed coats, and color of light. Lehmann's work (30, 31, 32, 33, 34), begun in 1909, continued through 191 5. Most of his experiments were conducted with seed of Verbascum Thapsus, V. thapsiforme, Epilobium roseum, and Ranunculus sceleratus. He showed the effects of substratum on germination in light, found the age of seeds to be an important factor, used Knop's nutrient solution as a stimulus instead of light, and found that salts favored germination of light-sensitive seeds in darkness. He claimed that light exerts its influence by starting or stopping some chemical changes in the seed, and established a relation between light and temperature. He also punctured seed coats as a substitute for light. Lehmann 252 BOTANICAL GAZETTE [april and Ottenwalder (36) experimented with Epilobium hirsulum and other seeds and showed that acid solutions and proteolytic enzymes can be substituted for light. They referred the light effect to activation of enzymes, but did little to prove their hypothesis. Pickeoltz (42) connected light effects and tempera- ture variations, and concluded that the influence of direct sunlight was mainly due to the heat rays which raised the temperature. Alternating temperatures helped the germination at different stages of maturity. In 191 2 a number of workers reported on the problem of light germination. Baar (i) investigated seeds of several Amarantaceae and found that most seeds of this family (Amaranthus, Celosia, and Blitum) have an aversion to light. He considered the age of seeds generally important for the occurrence and intensity of the light effect, and also related the light effect to substratum and temper- ature variations. Becker (2) brought forward a long list of examples of the light effect on germination of seeds. Haack (18) in his work on the Scotch pines demonstrated the influence of heat, and reported that temperature variations act as a stimulus to light-sensitive seeds, and that blue light is more favorable to germi- nation than darkness. Simon (47) reported that the salts of iron hindered germination of seeds in darkness, but increased it in light. Gassner (12) first reported on the germination of seeds of Chloris ciliata in 19 10. He found three factors which may be substituted for light, namely, increased oxygen supply, after-ripening, and high temperature. He claimed that light offsets the effect of the limiting factor, and showed that the chaff of Chloris ciliata prevents easy germination. His later work (14, 15) took up the action of chemicals. He considered the latent influence of light as related to seed bed, temperature, and after-ripening, the influence of light on germination, the influence of desiccation, and the relations between light and media favoring or hindering germination. From a tabulation of tests with seeds of different families he concluded that in these cases nitrogen, variously combined in the media, shows the same favorable action as light, but he included contradic- tory results. He considered the favorable effect of Knop's nutrient solution as due only to the nitrates present. He reported the seeds i 9 2i] GARDNER— GERMINATION 253 of Ranunculus sceleratus and Oenothera biennis as favorably influ- enced by light and by inorganic salts containing nitrogen, through a wide range of concentrations. Ottenw alder (40), working with Epilobiiim hirsutum seeds, found that the light requirement as regards intensity is closely related to temperature, the former increasing as the latter is lowered. The illumination period is related also to the temperature, but more closely to light intensity. Light-sensitive seeds are also favorably and strongly influenced by weak acids. The hypothesis of a catalytic influence of light is said to have received support from these observations. Materials A preliminary examination of 115 samples of seeds collected from Shaw's Gardens, the Botanical Gardens of the University of Michigan, waysides, swamps, and fields indicated the following as available for the study of the effect of light in germination: Daucus Carota, Nicotiana Tabacum, N. sylvestris, N. affinis, Nicotiana hybrids, Gentiana Saponaria, G. pannonica, Oenothera biennis, Verbascum Thapsus, Amaranthus caudatus, Rurnex crispus, Phora- dendron flavescens, and Datura Stramonium. Of those mentioned, the writer has been unable to find any previous report of light sensitiveness of seeds of Rumex crispus, Datura Stramonium, and Phoradendron flavescens. Of the light-sensitive seeds not previ- ously reported, seeds of Rumex crispus and Phoradendron flavescens are favored by light in germination, while seeds of Datura Stra- monium are inhibited from germinating by light, as will be shown later. Jonsson (23) in 1893 reported the seed of Daucus Carota as favored by light in germination. Nicotiana Tabacum seeds were first reported as light sensitive by Raciborski (43) in 1900. The seeds of Gentiana Saponaria, G. pannonica, Verbascum Thapsus, and Oenothera biennis were reported as light sensitive by Kinzel (24) in 1907. Baar (i) reported Amaranthus caudatus seeds as hindered in germination by light. The seeds of Gentiana are not conveniently suited to the purposes of this investigation on account of the longer incubation period. The seed of Amaranthus caudatus are not used because they are light-inhibited seeds. The seeds 254 BOTANICAL GAZETTE [april of Datura Stramonium have been found by careful experiments to be light inhibited and to require total darkness and a temperature of about 30 C. for germination. They are accordingly reserved for a future study. Detailed study of the germination of Phora- dendron flavescens seeds was deferred on account of the peculiar slimy ovary and the chlorophyll-bearing embryo. Seeds of Nico- tiana Tabacum, Rumex crispus, Oenothera biennis, Verbascum Thap- sus, and Daucus Carota were selected for this research because of their abundance and similar incubation periods. Germination in light and darkness Preliminary tests of Rumex crispus seeds on wet filter paper gave a germination of 84 per cent in light and 16 per cent in dark- ness after 8 days of incubation. TABLE I Treatment Percentage germination in light Percentage germination in darkness Cleaned, dried, and soaked in flowing water for 24 hours and incubated 16 24 12 6 Cleaned, dried, and incubated Cleaned, soaked for 4 days, and incubated 6 2 Preliminary tests of germination of seeds of Phoradendron flavescens, suggested by Wiesner's (51) results with seeds of Viscum album, are given in table I. Seeds prepared as indicated in table I were counted into Petri dishes containing wet filter paper as substratum and placed in light and total darkness to incubate at room temperature, which ranged from 18-25 C. The incubation period was 27 days. These results indicate that light favors the germination of seeds of Phoradendron flavescens. On account of the sticky nature of the pulpy ovary and the succulence of the single fleshy green embryo, it was almost impossible to remove the mass of enveloping material without leaving a favorable substratum for molds and bacteria on the one hand, and without injury to the embryo on the other hand. Moreover, with the best of care many of the seeds failed to germinate and became moldy. With these iQ-'i] GARDNER— GERM IN A TION 255 conditions we can understand the relatively low germination, and yet see that light favors the germination of these seeds. Seeds of Datura Stramonium were treated as shown in table II. Seeds were allowed to incubate for 17 days, and the results indicate clearly an inhibitory action of light on their germination. The TABLE II Treatment Percentage germination in light Percentage germination in darkness On soil, 26-30° C 6 2 O O 4 98 22 On sand, 26-30° C On filter paper, 20° C O On filter paper, 24° C 4 60 On filter paper, 30° C constituents of the soil solution seem to promote materially the germination of these seeds in darkness but not in light. Datura Stramonium seeds require different treatment from any of the other seeds tested, and accordingly are reserved for separate study. Light sensitiveness, after-ripening, and viability To establish standards for comparison with other data, and to indicate the degree of light sensitiveness, various light-sensitive seeds were incubated from time to time at room temperature (20-28 C.) on filter paper in light and darkness respectively. The results given in table III are fairly representative of these tests. These data indicate what may be expected of the various light- sensitive seeds under investigation when subjected to germinating conditions at room temperature 20-28 C. on wet filter paper in Petri dishes. It appears that the seeds of some kinds of tobacco are less light favored than others (3. Ntcotiana affinis, 108. N. affinis, 1. Nicotiana hybrid, and 117. Pennsylvania Havana tobacco). The results also indicate that not all of the seeds under investigation are entirely dependent on light for germination. A certain per- centage of each lot of Rumex crispus and Daucus Carota seeds usually germinates in darkness. It is also noteworthy that seeds of Oenothera biennis do not always germinate, even in light. The seeds under investigation seem to retain their light sensitiveness 256 BOTANICAL GAZETTE for long periods and to a rather high degree, especially those of Verbascum Thapsus and Nicotiana Tabacum. Attention should be called to low germination of newly harvested seeds of Oenothera biennis, Daucus Carota, and Rumex crispus. Tests for evidence of TABLE III Incubated 9-18-15 TO 9-29-15 Incubated 10-28-15 to "-5-15 Incubated 5-30-16 TO 6-6-16 Incubated 6-20-18 TO 6-28-18 Seeds Light Dark- ness Light Dark- ness Light Dark- ness Light Dark- ness Collected in 1914 i. Nicotiana hybrid . . . 3. Nicotiana affinis. . . . 8. Nicotiana hybrid.. . . 13. Nicotiana hybrid.. . . 22. Nicotiana hybrid.. . . 55. Verbascum Thapsus. 61. Daucus Carota 66. Oenothera biennis. . . 68. Rumex crispus 92. Nicotiana Tabacum. 96. Daucus Carota 9 1 79 48 84 55 81 63 4 39 77 1 56 57 1 1 19 2 8 1 87 87 58 89 73 80 78 76 88 65 45 5 2 10 10 18 87 74 74 89 61 87 7i 5i 75 90 60 43 35 6 20 4 31 4 84 82 67 80 55 53 32 60 30 45 19 13 7 1 52 6 Collected in 1915 97. Verbascum Thapsus. 98. Oenothera biennis. . . 99. Rumex crispus 100. Verbascum Thapsus. 101. Rumex crispus 94 2 36 74 61 1 94 48 82 73 94 45 63 94 4 1 1 82 61 80 39 92 44 61 79 75 75 49 81 100 86 1 40 2 11 11 24 71 2 66 60 30 105. Pennsylvania seed- iii. Connecticut seedleaf 117. Pennsylvania Ha- a period of after-ripening in Verbascum Thapsus were quite nega- tive. Newly harvested seeds of V. Thapsus germinate above 90 per cent in light and only about 2 per cent in darkness. Tests of still older seeds indicate that they retain light sensitiveness as long as they are viable. IQ2I] GA RDXER—GERMINA TION 257 Mechanical rupture In 1906 Crocker (7) succeeded in germinating a number of different kinds of seeds after breaking the seed coats. Kinzel (24) found that puncturing coats of some of his light sensitive seeds gave better germination in darkness. Gassner (13) found that rupture of the coats of seeds of Chloris ciliata permitted good germination in darkness at 34 C. Thus it seemed quite possible that the seeds under investigation might be brought to germination by such treatment. Accordingly a more carefully controlled experiment was made to determine the role of the several seed coats in germination. Seeds of each kind were rubbed on fine sandpaper and placed on moist filter paper in Petri dishes. The Petri dishes were carefully wrapped in black cloth and placed in a dark room at 24-3 o° C. for 8 days. Concurrently, sets of unabraded seeds were placed to germinate in light and darkness. TABLE IV Seeds Not abraded Abraded Light Darkness Darkness 60, 61 30, 16 75,32 io,39 60, 20 O, O 0, 1 0, 8 3, 27, 7 O, 1 40, 66* 6, . . 3, 5 25,15 Not abraded Darkness Xicotiana Tabacum . Rumex crispus Verbascum Thapsus . Oenothera biennis . . Daucus Carota o, 1 0, . . 1, 3 7, 1 21, 17 * Coats off. Mechanical abrasion of seed coats for various periods in rotating cylinders containing coarse quartz sand gave similar results. An examination of the data in table IV reveals the beneficial effect of abrasion of seed coats in but one instance. In the case of Rumex crispus abrasion of the seed coats yielded a percentage of germina- tion slightly exceeding that for light in the control, while the removal of the coats yields a percentage of germination even more than double that in light. This suggests that the seed coats of Rumex crispus inhibit or retard the entrance of some necessary factor, or perhaps retard the exit of some inhibiting factor, and that light in some way favors these movements. 258 BOTANICAL GAZETTE [APRIL Rupture by sulphuric acid As long ago as 1896 Rostrup (45) of the Danish Seed Con- trol found that concentrated sulphuric acid treatment hastened germination of hard seeds of Lathyrus syhestris. Todaro (50) used concentrated sulphuric acid on red clover seed with beneficial results. He also reported that various weed seeds, including those of Rumex crispus, were all destroyed by a brief immersion in sulphuric acid. Accordingly, to determine more certainly the role of seed coats in the germination of the five kinds of seeds, they were treated with concentrated sulphuric acid for periods previously determined, carefully washed in carbonate of soda solution, then in distilled water, and placed in germinators as previously described. TABLE V Treated with concentrated sulphuric acid Untreated Seeds Minutes in H,S0 4 Germinated in darkness In light 8 days In darkness 8 days (1) 10 days (2) 8 days (3) 8 days Nicotiana Tabacum Nicotiana Tabacum Verbascum Thapsus Verbascum Thapsus Daucus Carota O.S 1 I 0.5 1 6 8 8 10 O 23 1 27 62 38 1 O O O O 8 6 37 59 34 19 O O 17 41 23 42 42 72 72 31 ' 31 88 88 78 78 O O O 25 25 Daucus Carota Rumex crispus Rumex crispus O Oenothera biennis Oenothera biennis O Table V indicates that treatment of seeds of Rumex crispus and Oenothera biennis with concentrated sulphuric acid yields an increased percentage of germination in darkness. Treatment with concentrated sulphuric acid for longer or shorter periods than indicated gives no better germination of the seeds in darkness. In Daucus Carota there is apparently an injury. This experiment indicates that light acts on the coat of Rumex crispus seeds, and points in that direction in case of seed coats of Oenothera biennis. These results in the main agree with those of the experiment on abrasion of coats. They confirm the results with seeds of Rumex IQ2l] (/ 1 RDXER—GERM/.X. 1 TIOX 259 crispus and include the seeds of Oenothera biennis as being benefited in germination by acid treatment. Why the seeds of Oenothera biennis germinate better after treatment with H 2 S0 4 and not by abrasion is unexplained. Temperature and light Ottenwalder (40) claimed that within the range of tempera- tures which permit germination, light can be substituted for heat at low temperatures and heat for light at high temperatures. With Verbascum Thapsits and other seeds he found that germination occurred at high temperature in darkness and at low temperature TABLE VI Seeds Temperature centigrade 15° 19° 24 c 27° 30° 35° Light Xicotiana Tabacum. Verbascum Thapsus . Daucus Carota Oenothera biennis . . Rumex crispus Xicotiana Tabacum. Verbascum Thapsus . Daucus Carota Oenothera biennis . . Rumex crispus o o 12 O I 23 o 21 O 42 62 77 36 28 65 55 82 33 40 84 25 6 Darknes 11 6 2 o- 7 14 15 3 4 7 16 12 12 4 5 4 2 1 3 8 8 4 in light. In order to test this for American Verbascum Thapsus and to see whether it is generally true, the different seeds under investigation were placed to germinate in light and darkness at different temperatures. It was not possible to control closely temperature and prevent small fluctuations. These changes of temperature were never sudden, however, and had no effect except to increase slightly the percentage of germination in both light and darkness. The data reported represent results obtained from five different sets of determinations. Seeds were placed to germinate in Petri dishes on filter paper wetted with distilled water at tempera- tures indicated in table VI and allowed to incubate for 9 days. 260 BOTANICAL GAZETTE [april These data offer no evidence of a reciprocal relation between heat and light as suggested by Lehmann and Ottenw alder, not even in the case of seeds of Verbascum Thapsus, nor have any of the various tests indicated this reciprocal relation in the seeds. Indeed, in each kind of seed under investigation the optimum temperature for germination in light is very close to that for germination in darkness. In germination in darkness the results show rather definite minimum and maximum as well as optimum temperatures. It is especially noteworthy that high temperature and darkness did not induce germination of Verbascum Thapsus seeds, as claimed by Ottenw alder (40). No specific experiments were performed to determine the effect of light intensity on germina- tion, although early in this investigation it became a very familiar fact that very little illumination would induce germination. Good germination in darkness was frequently the occasion for repetition of an experiment, only to find that germination had been induced by leaks in the light screens. A comparison of table VI with data given elsewhere indicates that highest germination of light-sensitive seeds does not occur at constant temperature, but at temperatures fluctuating between 20 and 27 C. Effects of alternation of temperature, light, and darkness As long ago as 1882 Nobbe (39) and his students used alternating temperatures to promote germination, and in 1884 Liebenberg (35) referred light effects to variations of temperature in the germination of seeds of Poa pratensis. As recently as 191 1 Pickholtz (42) referred the action of light in promoting germination to the effects of heat rays. In an attempt to distinguish the effects of light from those of temperature the following experiments were performed. Seeds of each kind were counted into Petri dishes with filter paper wetted with distilled water as substratum. One lot of cultures was placed in darkness on February 9 at 40 C, where it remained for 17 days. Another lot of cultures was placed in dark- ness at temperatures ranging from o to 12 C. for 17 days. Another lot was kept in darkness and subjected alternately to high tempera- ture (40 C.) and low temperature (o-i2°C.) for nearly equal periods throughout the 17 days. The low temperature and the 1921] GARDNER GERMINATION 261 alternating temperature cultures were frozen on the morning of February 26. On this date observations were made and the cultures placed in light at room temperature to test viability. As shown in table VII, the constant high temperature effectively inhibited the germination of all seeds except those of Verbascum Thapsus. The subsequent incubation in light at room temperature showed fatal injury to the embryos of Daucus Carota, Oenothera biennis, and Nicotiana Tabacum. The constant low temperature delayed germination, but seemed to induce increased germination in light in seeds of Daucus Carota, Oenothera biennis, and Verbascum Thapsus. This is especially noticeable in Oenothera biennis seeds. TABLE VII Seeds Constantly at 40° C IN DARK- NESS FOR 17 DAYS (a) AND THEN AT ROOM TEMPERA- TURE IN LIGHT FOR 12 DAYS (6) Constantly at 0-i2° c in dark- ness for 17 days (a) AND THEN AT ROOM TEMPERA- TURE IN LIGHT FOR 12 DAYS (b) Alternately at 40 and 0-12 C IN DARKNESS FOR 17 DAYS (a) AND THEN AT ROOM TEMPERATURE IN LIGHT FOR 12 DAYS (b) Control at room tempera- ture for ii DAYS (a) (b) (a) (b) (a) (b) Light Dark- ness Verbascum Thapsus Rumex crispus Daucus Carota 14 O O O O 78 54 1 4 O O O O O 86 58 60 64 40 6 90 56 8 90 26 76 82 32 14 58 2 30 6 Oenothera biennis Nicotiana Tabacum The alternating high and low temperature treatment delayed the germination in the same way as did constant low temperature. As shown by the subsequent incubation, Daucus Carota seeds were injured most. The germination of seeds of Nicotiana Tabacum and Rumex crispus was materially reduced by this treatment, while the germination of seeds of Verbascum Thapsus was favored, and the germination of seeds of Oenothera biennis very greatly increased. In a further effort to distinguish effects of light and temperature an experiment was carried out as follows. Seeds were counted into Petri dishes, having filter paper wetted with distilled water for substratum, and placed under the following conditions: set a in light at io° C. for 8 days and then at 25 C. for 8 days; set b in darkness at io° C. for 8 days and then at 25 C. for 8 days; set c 262 BOTANICAL GAZETTE [APRIL in light at io° C. for 8 days and then in darkness at 25 C. for 8 days; set d in darkness at io° C. for 8 days, then at 40 C. for 4 days, followed by 4 days at 25 C. At the end of the 16 days' treatment all of the cultures were placed in light for 8 days at room tempera- ture. The experiment was begun July 24. Comparison of the data in a of table VIII with the control indicates that incubation in light at low temperature followed by incubation at room temperature results in reduction of percentage of germination of Daucus Carota seed. A comparison of a and the control indicates that alternating temperatures may in a measure replace light in the case of germination of Verbascum TABLE VIII 3* (a) (b) (e) tf) hUw Seeds < 10 < H M O W H •* §< « (J u •2 U °o U ££ rt a! -£°° "3 >> .Soo *J >> .So .Soo •^00 *^ rt 00 tuCT3 c-a c-a a u c-o fct-a c» rt >- co« 60 efl ^00 £«> £«> 73 «2 £«> 4J O ~00 J3 -c^-* ■^^2 ««« J 0) O >-> W « H H M H H « H H ^ H H VerbascumThapsus 94 O O 95 98 64 83 O 71 71 O I 88 Rumex crispus .... 8o 2 O 9« 98 2 63 O 94 97 O 31 34 Daucus Carota. . . . 60 4 O 14 i* 2 16 O 11 11 O 13 13 Oenothera biennis. 8s O 6q 6q 16 16 O 2 2 O 4 12 NicotianaTabacum 53 O 70 72 O 3 O 35 35 O 1 Thapsus seeds, that it is an important factor in the germination of Rumex crispus seeds, and further indicates the necessity of light in the early periods of incubation of Daucus Carota, Oenothera biennis, and Nicotiana Tabacum. A comparison of b with a points again to the necessity of light in Rumex crispus, Daucus Carota, Oenothera biennis, and Nicotiana Tabacum, and indicates that some inhibiting factor developed during the 8 days in darkness in the case of Oenothera biennis and Nicotiana Tabacum. A com- parison of c with the control indicates that light does its work on such seeds as Verbascum Thapsus, Rumex crispus, and in a measure on Nicotiana Tabacum even at low temperature, and that as soon as heat is supplied germination occurs. Incubating Daucus Carota and Oenothera biennis seeds at low temperature for a period of 8 iq.m] GARDNER GERMINATION 263 days, in light or darkness, produces a condition from which they do not recover when incubated at 25 C. in light or in darkness. A comparison of d with control a, b, and c indicates that sudden changes from extremes of temperature may delay germination of Verbascum Thapsus seeds, that such treatment inhibits the germina- tion of a large percentage of Rumex crispus seeds, and that it almost entirely inhibits the germination of seeds of Nicotiana Tabacum. The results in d confirm the observations on Daucus Carota and Oenothera biennis made in connection with b, namely, that some limiting factor develops during incubation in darkness at low temperature which is not easily overcome. The most noteworthy result of this treatment is the complete inhibition of germination of seeds of Xicotiana Tabacum. This is in agreement w T ith that found in b. Together these results when compared with the control indi- cate a light requirement for Xicotiana Tabacum seeds which is not replaced by any temperature combination tried. To summarize, this experiment shows that alternating tempera- ture may replace light in germination of Verbascum Thapsus seeds, that light is necessary for optimum germination of entire seeds of Rumex crispus, although change of temperature in a measure replaces light. The results of this experiment indicate that seeds of Oenothera biennis and Daucus Carota require light and medium temperature for optimum germination, and that incubation at low temperature in darkness permits a change which is not overcome by transfer to high temperature in darkness. Moreover, in Daucus Carota exposures to light at 25 C. did not bring about germination of these changed seeds. Incubation of Nicotiana Tabacum in darkness at io° C. did not result in increased percentage of germina- tion in darkness. Incubation of Ncotiana Tabacum seeds in light at io° C. promoted subsequent germination in darkness. Hot water treatment In a further attempt to induce germination in darkness, an* adaptation of the warm bath method of Molisch (37) was employed. The seeds were counted, w r rapped in filter paper, inclosed in little bags of cheesecloth, and plunged into hot distilled water for 0.25 minute and 0.5 minute respectively. Great care 264 BOTANICAL GAZETTE [APRIL was taken to plunge them promptly into cold distilled water, when the hot water was squeezed out. The seeds were then placed to germinate for 7 days at room temperature under the usual con- ditions. Table IX indicates what may be expected of hot water treatment of seeds. Treatment at lower temperatures was inef- fective and so was not tabulated. The experiment was begun March 4. The results of the warm bath treatment are mostly negative. The percentage of germination of Rumex crispus in darkness is TABLE IX Seeds t/3 M 2 < |2 Treatment at 90° c . ; germina- tion at room temperature FOR 7 DAYS Treatment at 75° c; germina- tion at room temperature FOR 7 DAYS Treatment at 6o°C; GERMINA- TION AT ROOM TEMPERATURE FOR 7 DAYS Untreated; germination AT ROOM TEMPERATURE FOR 7 DAYS Light Dark- ness Light Dark- ness Light Dark- ness < Light Dark- ness NicotianaTabacum NicotianaTabacum 0.25 0.5 0.25 0.5 0.25 0.5 0.25 0.5 0.25 0.5 4 22 22 O O 70 O 68 66 2 O O O O O 22 6 40 36 O 24 36 75 75 76 80 34 =;8 6 8 10 8 20 48 12 10 42 SO 20 40 67 65 40 28 58 86 II 34 3 8 20 15 8 20 35 28 62 2 Daucus Carota . . . Daucus Carota. . . 52 2 VerbascumThapsus VerbascumThapsus Oenothera biennis . Oenothera biennis . 78 O 50 6 Rumex crispus.. . . Rumex crispus. . . . 66 18 increased somewhat by treatment with hot water at 90 C, while that of Oenothera biennis is increased somewhat by treatment with hot water at 75 C. and 90 C. These results indicate the coat as the limiting factor in their germination. Treatment at ioo° C. for short periods might furnish interesting information. Water absorption To determine the relation of water absorption to germination in light and darkness, 2 to 3 gm. of each of the different kinds of seeds were weighed separately and placed under favorable con- ditions for germination. As soon as the first germination in light was observed, the seeds were dried rapidly and weighed carefully, and the percentage of water absorbed was computed on the dry 1921] GARDNER- -GERMINATION 265 weight basis. To confirm the results obtained a second series was treated similarly. Failing to obtain concordant results, two additional series of determinations were made. The variation in time of the appearance of the first hypocotyls and the uneven surfaces of the seed coats account for much of the variation in the amount of water absorbed. The results are given in table X. In view of the small size of the seeds, their irregular surfaces, the difficulty of uniform drying, and the increase of weight on the data of these determinations are not account of germination TABLE X Seeds Series i Percent- age of imbibed water No. of sprouts Series 2 Percent- age of imbibed water No. of sprouts Series 3 Percent- _ age of imbibed water No. of sprouts Light Series Percent- age of imbibed water No. of sprouts Nicotiana Tabacum. Verbascum Thapsus Daucus Carota Oenothera biennis . . Rumex crispus Nicotiana Tabacum . Verbascum Thapsus Daucus Carota Oenothera biennis . . Rumex crispus 92.9 13 82.3 10 108.5 3 65-5 93-3 12 58.8 2 199.0 25 93-3 H4-5 2 92.4 4 85-7 4 64.8 40.7 1 48.5 4 61.7 7 39-4 48.4 3 43-4 2 54-3 5 52.7 Darkness 60.0 50.0 67.7 1 63.6 81.2 68.0 90.0 2 76.9 90.7 46. i 1 97-3 1 64.0 45-i 108.5 1 49-4 1 49-9 53-i 8 52.4 1 48.9 1 45-8 surprisingly discordant. In some cases the high percentages of water absorbed is accounted for by the many and large seedlings which could not be removed without more seriously changing the data. After eHminating the cases open to suspicion on account of the numerous sprouts, imperfect drying, etc., there appears to be relatively little difference in the percentage of moisture absorbed by seeds germinated in light and darkness. In Nicotiana Tabacum seeds of series 4 the imbibition is 65.5 per cent with 8 sprouts in light, while in series 3 the imbibition is 67.7 per cent with one sprout in darkness, from which it appears that germination may occur even though a smaller percentage of water is absorbed. A 266 BOTANICAL GAZETTE comparison of determinations of absorption by Verbascum Thapsus seeds in light (series 2) and in darkness (series 4) indicates the same general relations. The data for the other seeds show similar results. From this experiment it appears that light is not necessary for the absorption of sufficient water for germination. Injection of seeds with water De Vries (8) , having abandoned variation of temperatures, high temperatures (4o-5o°C), and other treatments of seeds of Oenothera biennis as means of securing complete germination, injected soaked seeds with water under pressure of 6-8 atmospheres, after which he frequently secured germination.of 100 per cent. The TABLE XI Seeds Darkness FOR 17 DAYS Then in LIGHT FOR 7 DAYS Darkness for 15 DAYS Then in LrGHT FOR 7 DAYS Untreated • (a) (b) Light Darkness Verbascum Thapsus Daucus Carota Oenothera biennis Rumex crispus . . I 6 8 5 4 4 10 43 8 54 O 8 5 11 5 24 12 55 56 60 81 61 33 7i 83 O 21 8 Nicotiana Tabacum O seeds (table XI, a) accordingly were soaked overnight at a tempera- ture of 25-28 C, wrapped in filter paper, placed in water, exhausted of the air in their intercellular spaces by reducing the atmospheric pressure to 20 mm. for 1 hour, and then subjected to hydrogen gas pressure of 575-675 pounds per square inch for 24 hours. The seeds were then placed to germinate in darkness under the usual conditions. A second lot (b) was treated in the same way except that it was subjected to a pressure of 500-650 pounds per square inch for 48 hours. Both lots were germinated at room temperature. Evidently injection with water does not increase the germination of seeds of Oenothera biennis, Nicotiana Tabacum, Daucus Carota, or Rumex crispus in darkness. When the seeds are subsequently exposed to light, they germinate in one or both tests. These results confirm the conclusion arrived at in the weighing experiments, that impermeability to water is not the limiting factor in light germi- 19-21. G [RDNER— GERMINATION 267 nation. Perhaps better illumination of the injected Oenothera biennis seeds made possible the increased germination reported by De Vries. Increased oxygen supply In his investigation of the delayed germination of seeds of X ant hi ion, Crocker (7) found that the seed coat excludes oxygen, while Shull (46) found a very definite relation between the oxygen supply and the percentage of germination in seeds of X a lithium. In order to discover if increased oxygen supply would promote the germination of the light-sensitive seeds in darkness, the following experiment was performed. Counted seeds were placed on wet filter paper in open dishes and placed under water-sealed glass cylinders containing 40, 50, 60, 70, and 80 per cent oxygen respectively (table XII). Each cylinder was placed in a dark room at 23-28 C. and covered with a light-tight metallic cylinder. TABLE XII Seeds Percentage of germination in oxygen 40 50 60 70 80 Xicotiana Tabacum . Verbascum Thapsus. Daucus Carota Oenothera biennis. . . Rumex crispus 3 3 9 15 1 1 1 8 iQ iS A comparison of the germination in darkness in the presence of different percentages of oxygen shows an increase of germination of seeds of Daucus Carota and Rumex crispus with an increase of oxygen supply. Other conditions in each of the cylinders being the same so far as known, this must be attributed to increased oxygen supply. A similar experiment with higher and lower per- centages of oxygen would have been interesting, especially a test of germination in 20 per cent oxygen (ordinary air) under these conditions. It would probably have given results similar to those in 40 per cent oxygen and would have been rather more conclusive. The regularity of the increased percentage of germination, however, due to increased concentration of oxygen, indicates the reliability of the results. Clearly this experiment does not indicate an oxygen 268 BOTANICAL GAZETTE [APRIL deficiency in seeds of Nicotiana Tabacum, Verbascum Thapsus, and Oenothera biennis. Substrata Lehmann (30) reported increased germination in darkness of light-sensitive seeds such as Ranunculus sceleratus with soil as substratum. Baar (i) obtained an increased percentage of germination of seeds of Amaranihus when he substituted earth for filter paper as a substratum, but Ottenw alder (40), who used soil and sand as well as filter paper as substrata for his Epilobium hirsutum seeds, found beneficial results in his experiments with only one lot of sand. Investigation showed that the sand had been treated with acid which had not been thoroughly washed out. TABLE XIII Percentage of germination after 18 days On soil On sand In soil In sand On filter paper Seeds Light Dark- ness Light Dark- ness Light Light Light Dark- ness Verbascum Thapsus Daucus Carota 40 56 2 68 70 34 28 2 6 30 54 2 46 78 20 28 2 IO O 2 32 12 56 24 58 6 38 76 41 63 3 42 3 19 2 8 Oenothera biennis Rumex crispus Nicotiana Tabacum 7i 1 In view of the divergent results with the different seeds, it was deemed desirable to determine the relation of sand and soil to germination in darkness of these light-sensitive seeds. Also the question arose as to whether light was as necessary under natural conditions as under laboratory conditions for the germination of light-sensitive seeds. The substrata were carefully sterilized, uniformly wetted, and prepared for the seeds. The seeds in "sand" and in "soil" were buried to a depth of 0.25 inch. All were put under the same temperature conditions (23-2 6° C). The results are shown in table XIII. The experiment was begun May 13 and closed June 1. The germination of seeds of Verbascum Thapsus on soil and sand in darkness is somewhat higher than that on filter paper. The substratum appears to have exerted a slightly favorable effect on the germination of seeds of Daucus Carota, but none on the other i 9 2i] GARDNER— GERMINATION 269 seeds. Such results suggest a beneficial effect on some particular constituent contained only in seeds of Verbascum Thapsus. In light the percentage of germination seems to correspond roughly to the light intensity. Where the lighting is good, as on the filter paper or sand, the germination is good. Where it is diminished, as in the case of seeds buried in sand or soil, the germination is reduced. The increase of germination of seeds on sand or soil in darkness may be referred largely to the action of constituents of the soil and sand. The low germination of seeds of Oenothera biennis may be due to periodicity in dormancy, since seeds from the same lot gave a germination of 78 per cent in light and o per cent in darkness in October. From these results it appears that constitu- ents of soil may only partially substitute for light with some seeds and not at all with others. Effects of electrolytes The effects of electrolytes on the germination of light-sensitive seeds have been variously reported. Beneficial effects on germina- tion in darkness of Ranunculus sceleratus from hot water extracts of soils, Knop's nutrient solution, and salt solutions have been reported by Lehmann (30). He reported no benefit from cold water extracts of soils. Lehmann and Ottenw alder (36) found that weak acid solutions promote germination in darkness of seeds of Verbascum Thapsus, V. thapsiforme, and Lythrum Salicaria. Ottenw alder (40) reported that acids promote the germination of seeds of Epilobium hirsutum in darkness. Gassner (15) reported that nitrogen compounds such as nitrates, nitrites, and ammonium salts through a wide range of concentrations favor the germination of seeds of Chloris ciliata in darkness. As many of the electrolytes reported by these investigators belonged to the lyophobe or lyophile series, a systematic study of their effects was undertaken, to discover, if possible, some relation between electrolytes and germi- nation. Lots of 100 seeds each were counted into test tubes, about 2 cc. of a solution of an electrolyte added, and the tubes placed in darkness. After 24 hours most of the solution was drained from each test tube, which was promptly returned to the dark chamber for the seeds to germinate. The period allowed for germination was 7 days after soaking. The results are given in table XIV. 270 BOTANICAL GAZETTE [APRIL TABLE XIV Germination in darkness in various concentrations Seeds Z Z Z Z 8 d z 8 Z 8 d Seeds Z Z d 0.01 N 0.001 N Z 8 d z d Acetic acid Sodium iodide 8 77 2g 1 48 27 79 23 3 42 46 86 4 53 2.3 Verbascum Thapsus. . . 37 O 4 42 1 94 3 72 2 62 1 49 3 Daucus Carota Oenothera biennis Nicotiana Tabacum .... Nicotiana Tabacum . . . Verbascum Thapsus. . . Butyric acid Sulphuric acid 80 6 1 45 38 86 2 6 50 10 8 25 2 35 1 12 36 24 94 3 34 9 7 Nicotiana Tabacum . . . 2 Citric acid Potassium sulphate 6 38 36 40 11 10 61 35 95 8 5 52 54 98 13 12 68 58 87 1 44 12 89 58 5 100 1 1 38 8 Rumex crispus Daucus Carota Verbascum Thapsus. . . Rumex crispus Daucus Carota Oenothera biennis Nicotiana Tabacum . . . Tartaric acid Ammonium sulphate 7 2 21 14 7 52 59 81 10 13 75 50 53 8 67 27 51* 3 44* 36* 92 7 1 53 44 87 5 4 54 98 9 15 65 23 Malic acid Sodium sulphate 5 1 27 40 64 22 3 55 43 100 13 7 54 31 9i 6 1 60 83 91 2 1 58 4 94 6 48 9 88 2 41 10 Potassium sulphocyanate Lithium sulphate 80 8 70 3 75 2 4 65 I 88 2 2 61 7 :::: 93 3 2 46 17 3 53 12 90 1 37 9 Sodium sulphocyanate Nickel sulphate 2 63 93 4 52 53 14 63 1 3 i 33 2 62 2 28 40 13 86 21 3 7 9 Oenothera biennis * Little more than swelling. Injured. IQ2IJ a. i rdx i:r -germ in a tion 271 TABLE XIV— Coni inn ed Seeds % y 6 y 6 y 8 6 6 y 8 8 6 Seeds £ 6 b y 8 6 6 6 Zinc sulphate Cobalt nitrate Verbascum Thapsus. . . . 41 2 37 8 80 1 40 3 97 1 51 2 31* 1 4 45 13 99 4 2 57 15 93 2 8 16 10 Daucus Carota Daucus Carota Potassium nitrate Potassium hydroxide Verbascum Thapsus .... 36 3 8 55 50 82 2 18 61 23 95 8 55 23 84 6 7 44 17 100 6 9 55 34 98 9 4 42 53 Rumex crispus Oenothera biennis Nicotiana Tabacum .... Ammonium nitrate Ammonium hydroxide Verbascum Thapsus. . . . 62 3 70 54 100 14 66 73 82 n 1 68 34 93 8 1 64 28 I O O 31 1 90 I 15 95 1 3 56 12 Rumex crispus Daucus Carota Oenothera biennis Nicotiana Tabacum .... Sodium nitrate Sodium hydroxide Verbascum Thapsus .... 64 14 6 60 38 100 26 1 67 34 98 4 6 67 29 91 15 4 50 II 81 1 3 SO 21 ,6 2 II 57 3 83 2 6 16 Rumex crispus Daucus Carota Oenothera biennis Nicotiana Tabacum .... Aluminum nitrate . Hydrochloric acid Verbascum Thapsus .... 55 1 2 12 98 1 3 57 10 93 O 9 55 14 5 38 5 61 10 61 4 56 22 92 Rumex crispus Daucus Carota 2 Oenothera biennis 60 Nicotiana Tabacum .... 10 * Little more than swelling. From table XIV it appears that organic -acids, bases, and salts of monovalent, bivalent, and trivalent ions induce germination in darkness of seeds of Verbascum Thapsus (80-100 per cent), Oenothera biennis (40-60 per cent), and Nicotiana Tabacum (10-50 per cent), while they inhibit the germination of seeds of Rumex crispus and Daucus Carota. These results were confirmed in an attempt to determine the minimum effective concentration of the electrolytes. In this attempt it was found that as good germination in darkness 272 BOTANICAL GAZETTE [april occurs in ten-millionth normal solutions as in one-thousandth normal solutions. These results indicate no definite relation between the nature of the ion and germination. In another series of experiments on the relation of electrolytes to germination, with seeds from another crop, it was found that the germination of Verbascum Thapsus, Oenothera biennis, and Nicotiana Tabacum was inhibited, while the germination of Rumex crispus seeds was promoted in darkness by the action of the various electrolytes. This suggests that something in the conditions of growth, maturing, harvesting, or storage may have changed the sign of the charge of the ionizable constituents of the seeds. Further work on the effects of electro- lytes on the germination of these seeds is highly desirable. Soaking in solutions of electrolytes It is generally believed that forcing agents of germination such as light, enzymes, and electrolytes are most effective during the early stages of incubation. Kinzel (24) by soaking seeds of Nigella saliva in a solution of papayotin and asparagin for 5 hours and then in water for 24 hours secured a 30 per cent increase of germination of "light hard" seed. Ottenw alder (40) has reported that 24 hours is not sufficient time to secure the full effect of the acid on the germination of seed of Epilobium hirsutum, and that about 48 hours' soaking was necessary to get the best results from the action of the acid. An attempt was made, therefore, to determine whether soaking in solutions of electrolytes could pro- mote the germination of light-sensitive seeds. Seeds were soaked in the various solutions for 24-28 hours and washed in distilled water until all of the solution was removed. To avoid light effects, care was taken to work in very diffuse light. The seeds were spread on filter paper in Petri dishes and placed to germinate in light and darkness respectively, at room temperature, for 8 days. An examination of table XV A shows that soaking in rather strong solutions of hydrochloric acid promotes the germination in dark- ness of seeds of Nicotiana Tabacum, Verbascum Thapsus, Oenothera biennis, and Rumex crispus, while it hinders the germination of seeds of Daucus Carota in light. The beneficial effects of solutions I9-M GARDNER— GERM IN ATfON 273 of sulphuric acid appear only in the germination of seeds of Daucus Carota in darkness. Soaking seeds of Nicotiana Tabacum and TABLE XX A Seeds Light Darkness 0.1 N I 0.01 X 0.1N 0.01N i 00 Hydrochloric acid (soaked 26 hours) Xicotiana Tabacum. Verbascum Thapsus . Daucus Carota Oenothera biennis . . Rumex crispus Xicotiana Tabacum. Verbascum Thapsus Daucus Carota Oenothera biennis . . Rumex crispus Xicotiana Tabacum. Verbascum Thapsus . Daucus Carota Oenothera biennis . . Rumex crispus Xicotiana Tabacum. . Verbascum Thapsus . Daucus Carota Oenothera biennis . . , Rumex crispus 83 34 o 76 80 oS 57 56 5i 52 70 70 65 16 35 22 85 87 85 1 22 24 54 18 25 92 87 78 37 33 84 72 23 35 35 Sulphuric acid (soaked 28 hours) 76 79 72 83 69 76 87 66 5i 73 7i 81 61 62 7i 10 2 12 10 24 26 10 38 11 30 Sodium sulphocyanate (soaked 28 hours) 74 83 75 52 68 48 12 54 Sodium hydroxide (soaked 24 hours) 81 61 74 26 25 63 86 83 10 19 77 63 75 30 45 87 42 5i 22 12 59 76 68 17 26 21 o 21 8 31 19 5 26 12 24 19 5 26 12 24 TABLE XV B Percentage of hydrogen peroxide (soaked 27 hours) Seeds Light Darkness 5° 20 10 5 00 50 20 10 5 00 Xicotiana Tabacum 15 12 76 18 73 75 70 83 33 66 72 75 94 33 73 56 68 78 40 77 74 83 75 5i 68 6 26 43 38 48 5 36 81 6 61 12 70 46 2 53 10 39 19 Verbascum Thapsus Daucus Carota 06 Oenothera biennis 12 Rumex crispus 24 274 BOTANICAL GAZETTE [april Rumex crispus in solutions of sodium sulphocyanate appeared to promote their germination in darkness. Soaking in potassium sulphocyanate gave similar results. Soaking in solutions of hydro- gen peroxide promoted the germination of seeds of Nicotiana Tabacum, Daucus Carota, and Rumex crispus in darkness (table XV B). Germination in o.ooi N sodium hydroxide was about the same as in o.oi N. Soaking in solutions of sodium hydroxide gave increased germination in darkness of seeds of Verbascum Thapsus, Daucus Carota, and Oenothera biennis. These results were con- firmed by another set of tests. The reaction of the seeds to the different electrolytes indicates that the ions of the electrolytes are acting on different constituents of different seeds. While, as shown in the preceding experiment, the use of hydrochloric acid, sodium sulphocyanate, and hydrogen peroxide as substrata yields no increase of germination of Rumex crispus in darkness, this experiment shows that soaking for a short period (24-28 hours) in solutions of these electrolytes does pro- mote their germination in darkness. As Rumex crispus seeds were brought to germination in darkness by abrading and removing the coats, and by the action of concentrated sulphuric acid, their germination may naturally be referred to coat effects, the com- pounds acting on some constituent of the coat. The germination of Daucus Carota is not so easily accounted for. The germination in darkness was only slightly promoted by increased oxygen supply and on soil as substratum. The hydrogen peroxide may yield an increased oxygen supply and thus promote the germination of these seeds, but an explanation of the beneficial effects of sulphuric acid and sodium hydroxide on the same material is not easily made unless we refer to coat effects which have not been clearly indicated by other treatment. A longer period of soaking (48 hours) might have yielded data to settle this, as well as the failure of germination of the other seeds in certain solutions. Lipoid solvents Finding that lipoids occur in the coats and embryos of all the seeds and in the endosperm of four of them, it was thought desirable to determine the effect of acetone, alcohol, and ether on their 10 J I I GARDNER GERMINATION 275 germination. Seeds of each kind were soaked in acetone for 15, 30, and 60 minutes respectively, air dried for two hours, and placed under favorable conditions for germination in darkness. At the end of 8 clays of incubation (in darkness) the seeds were placed in light for 8 days, where none of them germinated. Other seeds were similarly treated with alcohol with similar results. A few seeds of Daucus Carota and Oenothera biennis survived the alcohol treatment and germinated in light and darkness. Other seeds were treated with ether, as indicated in table XVI. The results show no promotion of germination of light-sensitive seeds in darkness when treated with lipoid solvents, but rather show inhibition or diminution of subsequent germination in light. This is especially true for acetone and alcohol. Ether inhibited germina- TABLE XVI Seeds Soaked in ether 15 MINUTES Soaked in ether 30 minutes Soaked in ether 60 MINUTES Darkness Light Darkness Light Darkness Light Verbascum Thapsus Rumex crispus . . 15 O 28 35 17 76 43 O 17 30 3 17 74 64 12 O O 2 Daucus Carota 12 Oenothera biennis Nicotiana Tabacum 62 31 tion in darkness of all seeds except Daucus Carota, and diminished the subsequent germination in light of seeds of Verbascum Thapsus, Rumex crispus, and Nicotiana Tabacum. Ether treatment did not affect the subsequent germination of seeds of Oenothera biennis in light. Microchemistry In an attempt to find the substance responding to the action of light, an examination of the seeds was undertaken by micro- chemical methods suggested by Eckerson (9), and the nature and distribution of the different structural and nutritive materials were determined. Much of the information thus obtained has no evi- dent bearing on the problem of light germination and may best be presented in a separate publication. Some of the substances and conditions in these seeds which may function in light germination are fat, suberin, starch, and reaction. 276 BOTANICAL GAZETTE [april Starch occurs in the endosperm of seeds of Rumex crispus and Daucus Carota. It does not occur in the embryo of any seeds under investigation. Moreover, the hydrogen ion concentration is not likely to be materially changed by such hydrolysis of starch as may occur during their germination, and therefore we need not consider starch an important factor in their germination. Since suberin is found in the coats of Oenothera biennis and Daucus Carota, but not elsewhere, it can hardly be considered a common limiting factor in the germination of the light-sensitive seeds studied. Oily or fatty substances were found in the cell contents of coats, endosperms, and embryos of each of the seeds, appearing as small droplets of substance readily stained with Soudan III or Scharlach R. These lipoids almost never occurred as continuous layers which might obstruct the entrance of water or other substance necessary for germination, but usually as emulsions of fats in the cell sap. The results of the experiments on the absorption of water support the observation that there is no important obstruction of water by the constituents of the coats. Hence suberin and lipoids need no further consideration as limiting factors in absorption of water by these seeds. Using neutral red as an indicator, seeds soaked in water in light and in darkness, as well as dry seeds, were tested for the reaction of the different parts with results shown in table XVII. The outstanding result of the microchemical examination is the greater acidity of seeds incubated in light as compared with those incubated in darkness. This was found to be the case in each of the five kinds of light-sensitive seeds. The embryos incubated in light had a higher hydrogen ion concentration than those of the same kind incubated in darkness. This was especially noticeable in the hypocotyls. This result is contrary to Heinricher's (21) unsupported assumpton that the effectiveness of the fat splitting lipase was favored by the increased acid formation in darkness in Phacelia tanacetifolia. Moreover, Heinricher offered no experi- mental evidence of increased acid formation in darkness. Having all known external factors, except illumination, alike for the seeds under investigation, we may properly conclude that the varying factor, light, in some way brings about increased acidity of their embryos. 1921] GA RDNER—GERMINA TION TABLE XVII 277 Seeds Coats Endosperm Embryo Verbascum Thapsus Dry. . . Acid Acid . Acid Cell walls acid; con- tents neutral Cell walls acid; con- tents neutral Walls acid; contents acid Cell walls acid; con- Soaked ness . in dark- tents neutral Outer cell walls and con- Soaked in light . . tents alkaline to neu- tral; inner cell walls acid; contents alka- line Walls and contents acid Rumex crispus Dry. . . Acid Acid Acid Outer layer acid; cell contents neutral Outer layer acid; con- tents mostly acid Walls acid; contents acid Walls and contents neu- Soaked ness. in dark- tral Walls acid; contents Soaked in light . . neutral Walls and contents acid Daucus Carota Dry. . . Outer walls acid; inner alkaline Outer acid; inner alka- line Outer acid; inner alkaline Cell walls acid to alka- line; contents neu- tral to alkaline Walls acid to alkaline; contents neutral to alkaline Walls acid to alkaline; contents neutral Alkaline Soaked ness. in dark- Alkaline except at tip of hypocotyl Walls and contents acid Soaked in light . . Nicotiana Tabacum Dry. . . Acid Acid Acid Cell walls acid; con- tents neutral to alka r line Walls acid; contents alkaline to neutral Walls acid; contents neutral to acid Cell walls acid; con- Soaked ness. in dark- tents neutral to alka- line Cell walls acid; con- Soaked in light . . tents partly alkaline, partly neutral Walls acid; contents acid except at base of cotyledons 278 BOTANICAL GAZETTE TABLE XVII— Continued [APRIL Seeds Coats Endosperm Embryo Oenothera biennis Dry Partly acid Mostly acid Partly acid Slightly acid or neutral Slightly acid to neutral Neutral to acid Cell walls acid; con- Soaked in dark- ness Soaked in light . . tents neutral Cell walls acid to neu- tral; contents alkaline to neutral Walls and contents acid Quantitative determination of acidity To verify the results of the microchemical examination, 5 gm. of each kind of seed incubated in light for five days and 5 gm. of each kind incubated in darkness for five days were separately ground, digested in neutral alcohol and ether, and then titrated with N/10 NaOH. The results obtained were as follows: Seed Lijiht Darkness Rumex crispus 3 . 5 cc. 2 . 8 cc. Daucus Carota i .... 3. 3 2.8 Verbascum Thapsus 4.8 4.4 Nicotiana Tabacum 3.9 2.8 These results show greater acidity of seeds incubated in light than of those incubated in darkness, and confirm the findings of the microchemical examination. While the increase of titratable acidity was not large, it was measurable and repeatedly obtained, and apparently was sufficient in each instance to determine germina- tion. Since light is the variable factor in this and the preceding experiment, we may properly conclude that light initiates changes which produce the increased acidity of seeds incubated in light over those incubated in darkness. These results establish the fact that light functions in some way to bring about increased acidity in these light-sensitive seeds. There remains to show in the following experiment, if possible, how the acidity is increased. Effect of germination on substratum Having found that the embryos of these seeds become acid in reaction by incubation in light, it was thought that testing the IQ2IJ GARDNER— GERMIN. 1 TIOX 279 reaction of the substratum after a period of incubation might throw some light on what was happening in the seeds. A small quantity of each kind of seed was soaked in distilled water, 0.01N NH 4 N0 3 solution, and 0.01N NaN0 3 solution at room temperature, and the substratum tested for reaction with neutral red. The results are shown in table XVIII. TABLE XVIII Seeds Soaked 18 hours in distilled water Soaked 24 hours in 0.01X NH4NO3 Soaked 24 hours ix 0.01X NaN0 3 . 1 Light ! Darkness Light Darkness Light Darkness Verbascum Thapsus Alkaline Alkaline Alkaline Alkaline Alkaline Alkaline Rumex crispus Acid Acid Alkaline Xeutral Acid ;Acid Acid Alkaline Alkaline Alkaline Alkaline Xeutral Alkaline Alkaline Alkaline Alkaline Acid Daucus Carota Alkaline Alkaline Oenothera biennis Xeutral Xeutral Xicotiana Tabacum Alkaline From the results it appears that seeds of Verbascum Thapsus, Daucus Carota, and Nicotiana Tabacum excrete an alkaline sub- stance in darkness as well as in light; that seeds of Rumex crispus excrete an acid substance in darkness as w T ell as in light; and that seeds of Oenothera biennis excrete an alkaline substance in light. A quantitative experiment also verifies a part of these results. Weighed quantities of each of the five kinds of seeds were incubated in light for five days in tubes containing 2 cc. of 0.01 X NaN0 3 respectively. The results are given in table XIX. TABLE XIX Seeds Weight of seeds (gm.) Required to titrate Verbascum Thapsus . 7406 1.3385 0.6510 0.7580 . 8403 I . O CC. O . OI X HC1 Rumex crispus Daucus Carota 1.5 cc. . 01 X* XaOH o.q cc. 0.01 X HC1 ' 0.5 cc. 0.01 X HC1 Oenothera biennis Xicotiana Tabacum 1.8 cc. 0.01 X HC1 The longer period of incubation evidently allowed time for the excretion of a measurable amount of acid or base by each kind of seed. From these results it appears that seeds of Rumex crispus excrete measurable amounts of acid substance during incubation, while the other kinds excrete alkaline substances. 280 BOTANICAL GAZETTE [april Enzymes The favorable effects of light on the germination of seeds of Veronica peregrina in the early work of Heinricher (19) was referred to its effect upon chemical actions connected with the reactivation of reserve materials, and later (21) to its effect upon enzyme activity in the production and digestion of stored foods. He (22) referred the retarding effect of light on the germination of seeds of Phacelia tanacetifolia to its photochemical action on reserve materials, and assumed that the effectiveness of the fat splitting lipase was favored by the increased acid formation in darkness, while the irrefrangible light or the rays of the first half of the spectrum interfered, neutralizing the acid and thereby checking the decomposition of fat. It seemed possible that enzymes of some kind might be active agents, and light the stimulus or trigger in the germination of certain seeds. Just what kinds of enzymes function most in the germination of light-sensitive seeds has not been shown. To determine whether proteolytic enzymes were the important enzymes for the seeds, as Lehmann and Ottenw alder (36) believed for seed of Epilobiuni hirsutum, seeds of Verbascurn Thapsus and Nicotiana Tabacum were incubated in light and darkness respectively for four days and promptly ground in a little 50 per cent water solution of glycerine to which a crystal of thymol had been added. Small drops of extract from each kind of seed were put on nutrient gelatin according to the method of Giesen (16). After 30 minutes the extract was taken up with soft filter paper. There were very shallow pits formed where the extracts incubated in light had been. There were also shallow pits formed on the gelatin where the extract incubated in darkness had been. In fact, the pits produced by the extract germinated in darkness were deeper than those produced by the extract incubated in light. To verify these observations, the tests were repeated after allowing a more complete extraction of the enzyme. The extracts of seeds incubated in light were put in light, and the extracts of seeds incubated in darkness were put in darkness. The following day the same tests were repeated. Extracts from each lot of seeds were tested for their action by putting loopfuls on gelatin. After 30 minutes the extracts were removed separately, when it was i 9 2i] GARDNER— GERMINATION 281 found that well defined pits had been formed in the gelatin. More- over, where the extract was left on the gelatin for 24 hours, the pits became quite deep, even though there was abundance of thymol to inhibit bacterial action. These results were confirmed by the method of Gruss (17). Since the activity on gelatin of enzymes of seeds incubated in darkness was equal to or greater than that of seeds incubated in light, the favorable effect of light on germination of Nicotiana Tabacum and Verbascum Thapsus cannot be referred to activation of proteolytic enzymes. It has already been seen that starch does not occur in the embryos of any of these light-sensitive seeds, and that it occurs only in the endosperms of Daucus Carota and Rumex crispus. From these facts it is evident that hydrolysis of contained starch can increase the hydrogen ion of the embryos little if any. It has been seen that proteolytic enzymes develop equally well in darkness and light in these seeds, hence they can be rejected as important factors in determining light germination. Also, incubation in light does result in increased acidity of embryos over those incu- bated in darkness. It has been shown that the embryos of these seeds all contain fatty substances. The generally accepted method of demonstrating the presence of lipolytic enzymes is by the increase of acidity in the presence of fats. Inasmuch as development of acidity in the presence of light and fatty substance has been clearly demonstrated, it may be concluded that light activates the lipolytic enzyme to split the fatty substance to yield an acid. The results obtained with enzymes of seeds of Verbascum Thapsus and Nicotiana Tabacum do not support Heinricher's (22) assumption that light inhibited the action of lipase in seeds of Phacelia tdnacetifolia. On the other hand, the results indicate that light favored the action of lipase in seeds of Verbascum Thapsus and Nicotiana Tabacum. Discussion Coat effects The light relation of seeds of Rumex crispus is largely one affecting the coats, as is indicated by increased germination in darkness following abrasion and removal of coats, treatment with 282 BOTANICAL GAZETTE [april concentrated sulphuric acid, and increased oxygen pressure. Light may bring about some change in the coats of Rumex crispus to admit oxygen or other required substance, or permit the escape of some inhibiting substance such as an organic acid. It may change the relation of the lipoids from the oil water phase to the water oil phase, or break up a nearly continuous oil layer in the coat, thus allowing entrance or escape of some limiting factor. The presence of lipoids in the coats and the excretion of an acid instead of an alkaline substance during germination suggest that an enzyme acting in the coats hydrolyzes the lipoids, thus yielding acid and making the coats permeable to some required substance, or permitting the elimination of some inhibitory substance. There is some evidence of a coat effect in the germination of seeds of Oenothera biennis. While abrasion of the coats does not yield increased germination, hot water treatment and sulphuric acid treatment both yield considerable increases of germination in darkness. The presence of lipoids in the coats suggests the same explanation of the action of light as in the seeds of Rumex crispus, with the addition that the light may also have a beneficial effect on the constituents of the embryo. In the seeds of Nicotiana Tabacum, Verbascum Thapsus, and Daucus Carota there is little evidence of coat effects, there being no increased germination caused by abrasion, sulphuric acid treatment, hot water treatment, or increased oxygen pressure. The only results suggesting coat effects are increased germination of Daucus Carota and Nicotiana Tabacum when soaked in hydrogen peroxide. This increased germination might be referred to the effects on the embryos. The seeds of this investigation fall into three groups. The first is represented by the seeds of Rumex crispus, in which the coats must be made permeable to some external or internal substance by light, abrasion, or other agency before abundant germination occurs. The second group is represented by the seeds of Oenothera biennis, whose germination is partly dependent on the coats being made permeable, and partly on the activation of the embryos by light or chemical agencies. The third group is represented by seeds of Nicotiana Tabacum, Daucus Carota, and Verbascum Thapsus, i 9 2i] a 1 RDNER— GERMINATION 283 whose germination is not increased simply by making the coats permeable, but requires the action of light or a suitable substitute to induce good germination. MlCROCHEMISTRY The results of the various mechanical, physical, and chemical treatments of the light-sensitive seeds have offered few suggestions as to the nature of the effects of light on their constituents in inducing germination. The substitution of these various agencies for light has contributed little to an acceptable explanation of how light functions to bring about germination. These treatments, however, have served to localize the action of light and to determine the part of the seed affected. On the other hand, the microchemical examination yielded results which point to an acceptable explana- tion of the action of light on light-sensitive seeds. The outstanding findings of the microchemical studies were abundance of lipoids in each kind of seed and increased acidity of seeds incubated in light. Thus there are linked together light, lipoids, and increased acidity. Enzymes Since starch and other carbohydrates were not found in the embryos of these seeds and in the endosperms of but two of them, it is not necessary to give serious consideration to the probable reaction of the products of their hydrolysis. Moreover, since proteolytic enzymes were found to be equally active in light and darkness in Nicotiana Tabacum and Verbascum Thapsus seeds, they need not be considered as important causes of increased acidity of the seeds incubated in light. It remains to be considered whether the products of the hydrolysis of the lipoids are the cause of the increased acidity in light. The development of acidity in the watery extract of an oily seed like that of the castor bean is generally considered evidence of the presence of lipase. Such development of increased acidity in light was demonstrated quantitatively for four of the five kinds of seeds, thus confirming the results of the microchemical examination, and giving reasonable ground for inferring that lipase splits the fats thus yielding fatty acids in seeds germinated in light. 284 BOTANICAL GAZETTE [april Again, the presence of neutral or very faintly alkaline fats in the cells of the dry embryos and the development of acids in embryos incubated in light and no change or development of slight alkalinity when incubated in darkness is very significant. It is generally assumed that increased acidity of fatty substances indi- cates hydrolysis due to the action of enzymes. If this assumption be granted, the admission is necessary that light initiates processes which in some way result in increased acidity, which is followed by germination, and that where light is not admitted acidity does not develop sufficiently to cause good germination. Three explanations of how light acts may be offered: (1) light may act directly to split the fats to fatty acids and glycerine; (2) light may activate the lipolytic enzyme which splits the fats; (3) light may initiate some change that produces a little acid which may activate the lipolytic enzyme which splits the fats. While it is possible and even probable that light can act directly on the inclusions of cells to produce such changes as the formation of acid, it is hardly necessary to make this assumption. The proteolytic enzymes become active in the absence of light when the seeds are put under the usual conditions for germination. A certain percentage germinate in darkness even though they have not been treated with electrolytes or other stimulating agencies. Moreover, the amount of acid produced, though small, is relatively more than might be expected from the chemical action of light on lipoids. The evidence of the existence of lipase rather indicates either of the other explanations as much more acceptable. There is little choice between light activating the enzyme and light initiating some change which produces a little acid which may activate the lipolytic enzyme which splits fats. In either case light is the stimulus which initiates the changes leading to germination. Summary 1. The seeds of Rumex crispus, Datura Stramonium, and Phoradendron flavescens were found to be light sensitive. The germination of seeds of Rumex crispus and Phoradendron flavescens was promoted by light; the germination of seeds of Datura Stra- monium was hindered by light. i 9 ax] GARDNER— GERMINATION 285 2. Abrasion and removal of coats (ovary walls) of Rumex crispus seeds promoted their germination in darkness. 3. Treatment of seeds of Rumex crispus and Oenothera biennis with concentrated sulphuric acid caused an increase in the per- centage of germination in darkness. 4. No reciprocal relation between the effects of light and temper- ature was found. 5. Light was not necessary for the absorption of sufficient water for germination. 6. Injection of water did not yield increased germination in darkness. 7. Almost all kinds of single electrolytes, regardless of the nature of the ions, seemed to promote germination of seeds of Oenothera biennis, Nicotiana Tabacum, and Verbascum Thapsus in darkness. 8. Embryos of seeds incubated in light became more acid than those incubated in darkness. 9. Light seemed to activate lipolytic enzymes which hydrolyzed fats to fatty acids. 10. The germination of seeds of Rumex crispus in darkness was promoted (increased) by hot water treatment, abrasion, treatment with concentrated sulphuric acid, increased oxygen pressure, fluctuating temperatures, and soaking in solutions of hydrochloric acid, sodium sulphocyanate, and hydrogen peroxide. 1 1 . The germination of seeds of Nicotiana Tabacum in darkness was promoted by soaking in solutions of hydrochloric acid, sodium sulphocyanate, and hydrogen peroxide, as well as by the use of many single electrolytes as substrata. 12. The germination of seeds of Verbascum Thapsus in darkness was promoted by the action of light, fluctuation of temperature during incubation, alternating high and low temperatures, soil, and many single electrolytes as substrata. 13. The germination of seeds of Oenothera biennis in darkness was promoted during certain seasons by hot water treatment, sulphuric acid, preliminary incubation at low temperature, incuba- tion in alternating high and low temperatures, and single electro- lytes as substrata. 286 BOTANICAL GAZETTE [april 14. The germination of seeds of Daucus Carota in darkness was promoted by increased oxygen pressure and preliminary incubation at low temperature, while it was hindered by soaking in hydro- chloric acid and by the use of single electrolytes as substrata. The writer gratefully acknowledges the kindly advice and direc- tion of Professor F. C. Newcombe, in whose laboratory this investigation was begun; and also the valuable suggestions and criticisms of Professor William Crocker, under whose direction this investigation was completed. Alabama Polytechnic Institute Auburn, Ala. LITERATURE CITED 1. 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