IIIIII , I I I III II AN AWEMPT TO EXTRACT A $LOW‘ER- iNDUCING STIMULUS FROM CERTAIN PHOTOPERIOBICALLY SENSITIVE PLANTS I‘Imsis for The fiegm cf M. S. MICHIGAN WATEC” 113535: 133%.: [Xikd Emu Qv:(:‘zaf? If. any <‘ git I J ‘0- This is to certify that the thesis entitled presented b9 9 has been accepted towards fulfillment of the requirements for ' ' degree in I - Major professor J Date ‘ ‘31.’ 2n 0-169 AN ATTEMPT To EXTRACT A FLOWER-INDUCING STIMULUS FROM CERTAIN PHOTOPERIODICALLY SENSITIVE PLANTS By JAMES ARTHUR LOCKHART A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1951 THESIS ' IIlI fuiu‘ I II REVIEW OF THE LITERATURE OF PHOTOPERIODISM TABLE OF CONTENTS INTRODUCTION. . . . . . . . A. B. C. D. E. F. G. H. I. J. EARLY HISTORY. ; . . . . .'. . . . . . DISCOVERY OF PHOTOPERIODISM. CLASSIFICATION . . . . . . . INDUCTION AND INHIBITION . . PHOTOPERIOD AND ANATOMY. PERCEPTION AND LIGHT . O O NATURE AND TRANSLOCATION OF THE STIMULUS EFFECT OF TEMPERATURE. . . . . . . . . EFFECT CF OTHER ENVIRONMENTAL FACTORS. THEORIES ON THE MECHANISM OF PHOTO- PERIODISM. .I. . . . . . . . . . . . . III METHODSANDEATERIAIS........... IV VI VII A. EXPERIMENT 1, A Study of the Critical Per- B. EXPERIMENT 25 Attempts to Transfer the Flowering Stimulus in Gypsophila ele- 58.118.000.000 C. EXPERIMENT 3, Attempts to Transfer the Flowering Stimulus in Xanthium pennsxl— vanucum....... iod and the Induction Period in Gigs - philaelegans.............. RESULTS 0 O O ’ O O O O O O 0 DISCUSSION AND CONCLUSION . SUMMARY 0 O O O O O O O O 0 LITERATURE CITED. . . . . . 2N58K3457 O O .1 .5 .6 .8 IO 14 17 25 31 32 45 47 49 52 59 62 11 ACKNOWLEDGEMENT The author wishes to express his sincere appreciation to Dr. Charles L. Hamner under whose direction this stu- dy_was made. His suggestions and crit- icisms have been of great value and the writer is sincerely grateful for his guidance throughout the course of this 'Ork e I INTRODUCTION Until about the beginning of the present century it was generally believed that; since the genetic constitu- tion of the plant determines not only the type of repro- duction, but, also, to a considerable extent, the time of its onset, sexual reproduction in its entirety was under the control of the so-called "germ-plasm". The earlier hypothesis of Sachs (109), that hormones control organ formation lacked experimental evidence; and it was Klebs who took the early lead in the investigation of the physiological causes of flowering. Klebs showed that the environment, especially light and temperature, could greatly influence the transformation from vegeta- tive to reproductive growth (53, 55). He came to the conclusion that a balance between carbohydrates and min- erals, especially nitrogen, is the primary internal cause of flowering (54). Soon Kraus and Kraybill (59) reemphasized the prob- lem.of the relationship between carbohydrates and nitro- gen, and their work stimulated research which has con- tinued to the present day. In the subsequent studies, it has sometimes been forgotten that the results of Kraus and Kraybill's experiments with the tomato were based on fruit-set and not flowering. Although a great deal of work has been done on the relationships between the internal nutrients and metabolites of the plant and the subsequent growth responses, the definitive work on 2 this phase of the physiology of flowering remains to be done, in the opinion of this author. Shortly after the work of Kraus and.Kraybill came the discovery of an environmental factor with which the initiation of flowering in some plants could be quali- tatively controlled. This was the discovery by Garner and.Allard of the phenomena of photoperiodism. This presented for the first time a technique with which to study the physiological factors and the biochemical processes which are the direct cause of the flowering response. Rather slow progress has been made in the elucida- tion of the mechanisms involved in flowering however; partly because of the complexity of the problem and partly because the problem was not adequately defined until the work of Hamner and co-workers in the late thir- ties. They introduced into this field, for the first time, a point of view which produced immediate advances and it appears that their approach will be the one whidh will ultimately resolve the problem. II REVIEW OF THE LITERATURE EARLY'HISTORY Nearly all of the early work relating to the influ- ence on plants of the daily duration of light was direc- ted toward determining the extent to waich both the growth and the deveIOpment of the plant may be stimula- ted by lengthening the daily light period. Considerable prominence was given the question of whether plants would thrive under continuous light or if they need a daily rest period. According to Smith (112), apparently the first ref- erence in the literature to the influence of length of day on plants is found in Carl von Linne's "Ron omvax- ters plantering grundat, pa naturen" (641, published in 1739. However, Linne ascribes the rapid growth and ear- 1y maturity attained by plants in polar regions to addi- tional heat supplied by the continuous sunlight, rather than the additional light as such. Schubeler (110) in 1879, advanced the idea that the cereals and other spe- cies of plants, when gradually transferred from lower to higher latitudes, undergo definite changes in growth characteristics, and ascribed the observed effects to direct or indirect action of additional sunlight. Sie- mens (111) in 1881, reported results of growing plants under eleCtric-arc lamp to replace or supplement sun- light and he reached the conclusions that, under suit- able conditions it can replace sunlight, and the plants apparently do not require a daily rest period. KJellman (52) during an expedition to the north coast of Siberia in 1878 and 1879, conducted experiments with the arctic species Catabrosa algida and Cochlearia feulestrata which.were exposed to continuous northern light or a twelve hour day. As Opposed to the shortened photoperiod the continuous light resulted in a more rap- id growth and earlier and more profuse flowering. The effects on flowering, however, were only quantitative and were not particularly striking. In the period 1891-1895, Bailey (5) conducted in- vestigations with light from arc lamps used for a por- tion or all of the night as a supplement to daylight, particularly with the idea of forcing vegetables. The additional light hastened the growth of lettuce and in- duced early flowering in spinach. Rane (99), working along similar lines with the incandescent carbon fila- ment lamp, obtained much.the same results with lettuce and spinach and observed earlier blooming in certain flowering plants. Corbett (28) demonstrated that night Illumination, as a supplement to daylight, markedly stimulated tOp growth at the expense of the roots, in sugar beets; and he observed stimulation of growth in several other plants. According to H. A. Allard (1) an interesting ref- erence to the photOperiod as effecting plants is given by A. Henfrey, in his book, “The Vegetation of Eur0pe", . 1852, where he proposes that the length of day is a I factor in the natural distribution of plants. Klebs also, as early as 1913, seems to have sensed the fact that photOperiod affected the time of flowering of some of his experimental plants. With.ggmpervivium funkii he was not able to secure flower development in the winter by changes in temperature, nutrition, etc., so he exposed them to a few days of continuous electric illumination. They produced flowers in the same green- house in which Other, non-lighted plants remained vegeta- tive. Concluded Klebs (54): "In der freier Natur wird sehr wehrscheinlich die Bluteseit dadurch bestimmt dars vonder Tag und nacht- gleishe (21 Marz) ab die Lange des Tag es Zunimmt, die von einer gewissen Dauer ab die Aulagen der Blute veran- 1asst. Das licht wirkt wohl nicht als ernah render Factor, sondern mehr Katalytisch." In the work of Tournois (120) with Cannabis sativa and Humulus lgpanicus, published in 1912, there is found what is apparently the first definite suggestion that the attainment of the flowering stage may be hastened by a relatively short photOperiod. Tournois demonstrated that a precocious type of flowering which occurs in very early spring plantings of these species can be repro- duced by allowing the plants to receive sunlight for on— ly six hours daily. Apparently he did not extend his re- searches in this direction. DISCOVERY OF PHOTOPERIOD To Garner and Allard, however, goes all the credit for demonstrating the fact that the length of day quali- tatively controls the change from vegetative to repro- ductive growth in.many plants (34). It was a big step, from the point of view of that time, to accept the fact that such a "dilute" factor as the length of daylight should have Such.a marked influence on deveIOpment, but from the first report this conclusion was inescapable. Their discovery came as a result of breeding exper- iments with tobacco, in which a new variety, "Maryland Mammoth", failed to flower during the summer months, and of Soybean experiments in which successive plant- ings throughout the spring and early summer all tended to flower at the same time. In the case of the tobacco the investigators were at firdt misled by the fact that plants in small pots in the greenhouse during the winter and early spring flowered profusely, suggesting a nutri- tion angle. After a time, however, the critical obser- vation showed that as spring approached, from the stumps of the flowering plants there arose new shoots of typi- cal vegetative growth. From this it seemed quite clear that some seasonal variation was involved. Experiments soon showed that it was the duration of the light period which regulated the reproductive habit. They made many subsequent studies (35, 36, 37) to determdne the number of plants which are photOperiOdi- cally sensitive. Particular attention was paid to the reproductive reaponse but the effects of photoperiod on tuberization, bulbing, character and extent of branch- ing, root growth, pubescence, pigment formation, abscis- sion and leaf fall, dormancy and death were also stud- ied. They observed that other environmental factors, particularly temperature, modified the effects of photo- period. CLASSIFICATION Garner and Allard (34) prOposed the classification of plants in relation to photoperiod essentially as it stands today, because they early recognized the impor- tance of the length of the dark period. They called those plants "short-day plants” which have a maximum critical photOperiod, above which.flower formation is inhibited (i.e. a minimum.dark period requirement). These plants which have a minimum.critical light period requirement (limited dark period tolerance) were called "long-day plants"; while plants whose flowering response is not qualitatively affected by daylongth.were called "indeterminate". The parenthetical expressions above indicate more Closely the present accepted definitions, especially for short-day plants. In the case of long- day plants a more complicated situation exists; they will flower on light periods greater than their criti- cal, regardless of the length of the dark period, but they will also flower on short cycles of light providing the dark periods are also short (e.g. six hours light-- six hours darkness; five seconds light--five seconds darkness) (37). Within these categories there is the greatest pos- sible variation. Some short-day plants may remain vege- tative almost indefinitely if kept on a long-day, while others will eventually flower, and in some the short-day character is only indicated by the formation of flowers on nodes nearer the base of the plant (11). The long- day plants also intergrade imperceptibly into the indeter- minate, there being plants in which a long-day may speed flower formation by only a few days. lO INDUCTION AND INHIBITION Early in their studies Garner and Allard (35) observed that by exposing certain plants to the preper photoper- iod for a comparatively short length of time (in Biloxi soybean for ten days) flower and seed production would subsequently occur. Since then numerous investigators have observed similar phenomena, although considerable variation is reported (18, 19, 31, 66, 67, 68, 98). Rasumov studied after-effects in representative long- and short-day plants (100), and concluded that treatments early in the life of the plant would marked- 1y effect the subsequent growth and development of the plant. In a rather striking demonstration, Murneek ‘ started plants of Rudbeckia in the greenhouse under nat- ural light so that as the season progressed the day- length.increased from ten to thirteen hours. At the longest daylongth.some of the plants were moved to a long-day bench where they formed stalks and flowered normally; of the others which at the same time were transferred to a short-day bench: some remained vege- tative, some formed I'vegetative flowers" and some formed true flowers on the rosettes. The different reactions, Murneek suggested, could be the result of different de- grees of development in the individual plants at the thme they were exposed to the long-day (13 hour) photo- periods. Those which became I'ripe to flower" first re- ceived the longest induction, enough to induce flowering, 11 although not stem elongation; those that remained vege- tative had received little or no induction; while those which produced I'vegetative flowers" had been subjected to an intermediate induction period (75). A variation of this explanation is also possible: it may be that the critical period varies in the individual plant (sug- gested by work reported here, see RESULTS), then some would have received a longer induction period than others. The phenomena of induction exhibits striking varia- tions between species of plants, when viewed from.the point of view of the stimulus produced. Induction is most striking in short-day plants; cocklebur requires only one short-day (at normal temperatures) for induc- tion (48), and Biloxi soybean only two days (12). Long (65) has shown that in the soybean these two cycles must be consecutive, that regardless of the number of cycles given on alternate days no flowering would oc- cur. Hamner has demonstrated that twenty hours of light between the subsequent dark periods is the maximum.inter-v ruption which will still permit induction to occur in the soybean (44). In cocklebur the number of cycles required to induce flowering can be increased to about seven by lowering the temperature during the dark period to 40° F. In this case it has been shown that the stimulus will ear- ry over one long-day after the first four induction cy- cles (44). This suggests that the stimulus produced during each dark period rapidly disappears, yet Hamner‘ and Bonner (48) found that, in cocklebur, the stimulus 12 continues to be produced after the leaf is transferred back to a long-day, although this is not true of Biloxi soybeans (44). Hamner and Bonner concluded that once the reaction had attained a certain rate, it became autocatylytic in cocklebur. In an annual beet (a long-day plant), which has an induction period of about twenty days, it has been demon- strated that, after the first ten days, it is possible to interrupt the induction cycles with short-days for more than ten days without any apparent reduction in the quantity of the stimulus (80). The destruction or accumulation of the stimulus has not yet been studied in other plants but it is already clear that a great diversity of response exists. It seems likely (but not certain) that this diversity repre- sents differences in the rate of disappearance of the stimulus. It would seem possible to test the validity of this assumption, and if it is found to be true, to determine whether this represents a difference in the nature of the stimulus or differences in the internal physical and chemical environment of the plant to which the stimulus is exposed. Murneek has repeated the demonstration of the in- duction of flower formation without stem.elongation in Rudbeckia with high temperatures (78). He considers the effect of the short-day on long-day plants to be in- hibitory. Greulach (43) confirmed the findings of Mur- neek, and he too considered the effect of short-day on 13 the elongation and flowering of long-day plants to be an inhibition of flowering. Neither, apparently, ser- iously considered the alternative; that elongation and flowering take place only in the presence of some stimu- lus, and when this is withdrawn (as in short-day condi- tions) the plant reverts to vegetative growth. The question remains in doubt, although recent work On the action spectra of photoperiodism.seems to support their conclusion (see LIGHT AND PERCEPTION). 14 PHOTOPERIOD AND ANATOMY In 1936, Roberts and Wilton (107) reported that plants induced to flower, in comparisOn to those not in- duced, showed more complete differentiation of the xylem and phloem, and assumed that this meant a cessation of cambial activity. However, Psarev reported an increase in stem diameter of the soybean at the time of flower- ing (97). Most of the subsequent work has been done by Roberts and his associates; they repeat and refine their previous observations. The rosette type Of growth, that is, the growth and develOpment of many leaves with no corresponding elonga- tion of the stem, is characteristic of many long-day plants when grown under short-day conditions. Upon exposure to a period of photoinductive cycles, the stem elongates and flowers are formed at the apex. In the case of Gypsophila ele ans, reported in this paper, the elongation takes place almost equally at all internodes, so that little or no indication of the previous rosette condition remains. In other plants the same general habit may be observed or the stem.may elongate only from the apex, giving rise to a scape with few or no leaves. Roberts and his associates, in various papers (103, 105, 106, 107, 124, 125) have reported a cessation of cambial activity in all types of plants at the time of flower induction. They c1aim.to have observed this phe- nomena in typical leng-day plants, but nowhere do they \ 15 mention or even imply that their noneflowering long-day plants were in the rosette condition. 0n the contrary, the only mention made of the rosette habit of growth, in Robert's papers, appeared when he stated (106): "An extensive study of the relation of stem anatomy to flow ering of nonocots has not been attempted as so few of the commonly available species have stems suitable for sampling when in a non-flowering condition. The grains and grasses as well as most other locally grown species of this group are in an induced state before stems long enough for sampling of internodes are produced." This certainly implies that long-day plants with the rOsette habit of growth either were not used or were used only after they had begun to elongate. A number of examples are given, including a few presumed long-day plants, but this writer has been unable, as yet, to de- termine whether any of those mentioned usually grow with the rosette habit under short-day conditions. Most of the "long-day plants" which they used do not show parti- cularly striking long-day responses. While all long-day plants do not grow as rosettes and all plants with the rosette habit are not typical long-day plants, never-the- less the two characteristics are strongly correlated. It is certainly reasonable that determinate annual plants, upon reaching a late stage of maturity and fruit- ing, would lose cambial activity. As the plant soon dies it is apparent that this must occur. In plants of a rosette habit, however, a great deal of cell elongation 16 must occur; and preliminary observations by the author bear out the logical assumption that considerable cell division also occurs. Even Robert's own evidence fails to bear out his conclusions. He shows many micrOphotographs comparing cross-sections of flowering and non-flowering stems (106, 107, 116), and these show clearly that in flowering stems differentiation Ofxylem and phloem.cells occurs much.more rapidly; lignification Of the xylem can be ob- served immediately adjacent to the cambium region, while in non-flowering stems several differentiating cells separate the lignified.xylem cells from the phloem. This, however, does not necessarily prove that cambial activity has ceased. It could be preposed rather that the subse- quent differentiation was accelerated. In many of his photographs the number of vascular layers in the flower- ing stem appear to greatly exceed that of the non-flow- ering stems. 17 PERCEPTION AND LIGHT Garner and Allard (35) found, by exposing only a portion of the plant to aninductive photOperiod, that in Cosmos the effects are largely localized; that por- tion of the plant which received the inductive cycle responded typically but the remainder of the plant re- mained uneffected by the treatment. They pointed out that the inductive stimulus may, in some cases at least, be transported from one part of the plant to the other, for example, in the tuberization of potatoes. This was later verified with several other tuber producing plants by Rasumov (101, 102). Knott (56), in extensive work with the spinach, was led to believe that the leaves in some way hasten the photOperiodic effect in plants. In 1936 Cailah- jan (20), Moskov (72), and Psarev (96), apparently working independently, each came to the conclusion that the green leaves are the organs which receive the I'photOperiodic stimulus", and that certain physiologi- Cal processes arise there which act on the growing points, "directing" them either to further vegetative growth or to floral initiation. CailahJan and Moskov preposed substances of a hormonal nature, which Cailah- Jan called "florigen". At the same time Kiupper and Weirsum (60) in Holland advanced the same idea. La- ter investigators, including Hamner and Bonner (48) and a great many others, have confirmed the fact that 18 the leaf is the organ of~perception of the photoperiodic stimulus. Borthwick and Parker (13) found that the youngest fully expanded leaf was the meat effective in the recep- tion of the stimulus in Biloxi soybean and this has been found generally true of other species (46). Considerable early work was attempted on the action spectrum of photOperiOdism (126) but the technical prob- lems involved were numerous and it was not until the work of Parker, Hendricks, Borthwick and Scully (14, 92, 939 that clear-cut results were obtained. By inter- rupting the dark period in short-day plants and extend- ing the light period in long-day plants with light of very narrow wave lengths they were able to accurately determine the action spectrum for these plants. They found that the spectra for the two types is apparently identical, and that wlile the spectra generally resemp bled that of the chlorOphylls, it seems quite clear that the differences found were real. This was further borne out when it was found that this spectrum coin- cided very closely with the spectrum for the so-called I'etiolation effect" in peas (94). In both these re- Spouses there is a comparatively high effect in the red- and low activity in the blue portion of the spectrum. A similar action spectrum.has been Obtained by Weintraub and Price (122) for the inhibition Of elongation of the first internode of A1335. Goodwin and Owens (39) re- ported similar results and they attributed this toy” 19 absorption by protochlorOphyll masked in the blue by carotenoids. Parker and Borthwick (91) have pointed out that this action spectrum does not fit that for the transformation Of protochlorOphyll to chlorOphyll, and they suggest instead the resemblance of these spectra to the absorption spectrum of the pigment phycocyanin. At this time, however, the receptor pigment remains un- identified. This light effect is the one which interrupts the dark reaction in short-day plants, and apparently inter- rupts a dark reaction in long-day plants as well. It may be characterized as being fully effective even when of short duration (1-30 minutes) and at very low inten- sities (1-10 f.c.). * In both long-day and short-day plants light of "normal" intensities is required for at least a portion Of the day. There is strong evidence that this high intensity light effect is involved in the production of the precursor of the stimulus (44) and it may play a significant role in transport also. In contrast to the low intensity light effect (discussed above) the high intensity light effect is apparently photOSynthe- tic in nature (see THEORIES). NATURE AND TRANSLOCATION OF THE STIMULUS It was pointed out earlier in this paper (see PER— CEPTION AND LIGHT) that the leaf has been shown to be the organ of perception of the photOperiodic stimulus. It therefore seemed likely that a study of the translo- cation of the stimulus from the leaf to the growing point presented a point of attack in studying the nature of the stimulus. . Investigations in this direction have shown that the transmission of the stimulus apparently takes place only through living cells, as indicated by the fact that its movement out of the leaf can be inhibited by low temperatures (15, 24), scalding (127), girdling (as), and narcotics (24). Further work (20, 21, 22, vs) demenstrated that the stimulus could readily cross graft unions. This work showed that induced plants, when grafted to non-induced plants of the same species, could transmit the stimulus to the non-induced plants causing them to flower. It was also demonstrated that when short-day plants were grafted to indeterminate plants (both kept on a long-day) the short-day plants were induced to flower, indicating that the stimulus in these two types are similar. Some of these authors also reported the cross-transmission of the stimulus between short-day and long-day types but this has not been confirmed since the early work. Hamner and Bonner (48) reported that the stimulus 21 could cross a "diffusion contact" prepared by separat- ing the graft partners with a piece of lens paper, and careful micrOSCOpic examination of the graft, made after the termination of contact, revealed no evidence of tis- sue contact. However, Withrow and Withrow (127) in at- tempting to repeat these experiments failed to effect transmission except in those cases when subsequent ex- amination revealed tissue contact, if only a few cells. In another attempt to demonstrate transfer of the flow- ering stimulus across a non-living contact, Moskov (72) reported that by placing an induced leaf in close prox- imity to the cut petiole of a non-induced plant in a water medium, induction would occur. Galston attempted to repeat this experiment, but without success (33), and Melchers and Lang have also reported failure of the stimulus to cross a I‘diffusion contact" (70). Hamner and Bonner (48) demonstrated that while an induction cycle of one day sufficed to induce flowering in the cocklebur, the leaf must remain on the plant for at least four days for flowering to subsequently occur. They also present evidence to show that the leaf contin- ues the production of the flowering stimulus after it has been returned to a non-inductive cycle. Long (65), as was mentioned above, found that a short inductive cycle given to Biloxi soybean resulted in flowering at only a few nodes in the vicinity of the induced leaves. Hamner and Bonner (48) showed that the non-induced leaves influence the translocation of the stimulus; when~two- branched cocklebur plants were treated so that one branch (the donor) received an inductive cycle while the other (the receptor) remained on a long-day the recep- tor flowered, but this could be prevented if the young leaves of the receptor were removed. They also demon- strated that if the older leaves of the receptor branch were also removed, flowering would occur. This strongly suggests that the Old leaves which are net induced exert an inhibitory effect on the trans— location of the stimulus, while the young leaves pro- mote this translocation. This was confirmed.by Borth- wick and Parker (11) who fOund that only by defoliating the leaves of the receptor branch of a two-branched soy- bean could the receptor be made to flower, and by Heinze, et a1., (51) who showed that this is also true of grafted soybeans. Moskov (74) reported that if the old leaves are kept in complete darkness instead of on a long-day their inhibitory effect was decreased. Stout (115) has combined these effects in an experiment in whiCh.annual beets having three stems were treated so that one stem was on a long-day, another was on a short-day and the third was kept in continuous darkness. The stems in long-day and continuous darkness flowered (in that order), but the stem on a short-day remained vegetative. Stout concluded that the stimulus moves with.the carbohydrates. Although a definite conclusion hardly seemm warranted as yet, this remains one of the most likely possibilities. Recent experiments by Roberts- 23 (104) in which he shows that, when the induced leaves of cocklebur are shaded, the plants respond much.more slowly than the controls but sucrose sprayed on the leaves of the shaded plants will overcome this delay, lends further support to this theory, although Roberts draws an entirely different conclusion from his work. The buds also exert an effect on the translocation Of the stimulus; if they are removed from the I'donor" branch of cocklebur, the stimulus appears to reach the receptor with greater force (48). Several possible explanatiOns suggest themselves to account for these phenomena. If, as Stout has sug- gested, the stimulus is translocated with the carbohy- drates it would be possible to visualize the effects exerted by the young and old leaves of the receptor. It has been demonstrated that the growth regulator 2,4D is translocated in this manner (71, 121). With this hypothesis it would be more difficult to explain the response arising from the removal of the buds, but it could be suggested that this would remove a demand.for sugars on the donor side. The effect of the removal Of most of the buds on the receptor side might furnish a clue to this question. The fact that these organs are the major centers of auxin production in the plant also suggests a possible relationship, although Bonner (6) has shown that the primary effect Of applied auxin in inhibiting flowering of Xanthium is on the production 24 or the translocation out of the leaves., It could also be suggested that the buds use up some Of the stimulus which.would otherwise be free to move to the receptor, and, not necessarily in conjunction, the Old leaves might absorb or destroy that portion which, in some way, comes under their influence. It should be simple to resolve this problem. 25 EFFECT OF TEMPERATURE It'was believed for a time by many workers that in photoperiodically responsive plants, the duration of light was the only factor which affected the flowering response. Soon, however, Thompson and others discov- ered that celery (118), beets (117,) lettuce (119), and stocks (95), for example, which give responses to photo- period, may also be induced to flower by chilling. Chroboczek (27) working with beets, showed that favorable conditions of light and temperature are es- sential to the develOpment of a fertile inflorescence, as well as to initiate flower primordia. By regulating the temperature and the photOperiod, the time normally required for seedstalk formation could be greatly short- ened or materially lengthened. Steinberg and Allard (114) reported that the critical period for flowering in sOybean, Rudbeckia bicolor, and beet may be altered to a limited extent by temperature, and, conversely, the favorable temperature range for flowering may be shifted by the action of the photOperiod. Knott (57), studying the effect of temperature on the photOperiOdic response of spinach, found that with photoperiod fixed at fifteen hours per day, seed-stalk elongation occured sooner if the temperature during the treatment was held at 600 to 700 F., than at a higher or lower tempera- ture. Roberts and Struckmeyer found that the responses of many species of plants to photOperiod could be 26 materially altered by varying the temperature above or below normal (105). Gilbert (38) studied the interre- lation of length of day and temperature in Xanthium ppnnsylvanicum, and reported that temperature influ- enced the time of production of flower primordia. It is not clear in many of these studies whether the temp perature exerts its primary effect on the initiation of flower primordia or on the subsequent visible flower- ing response of the plant. In the strawberry, the production of flowers and runners are independent functions Of photoperiod and temperature (30). Flowers, which are produced under short-day conditions, can be induced on a longer photo— period at lower temperatures. The production of runners, a long-day response, is encouraged by high temperatures. In the onion a very interesting situation exists (50).. Bulbing and flower-formation are antagonistic. Bulbing is a response to long photOperiod and is irre- versible after an adequate induction period, except that the tendency may be destroyed by high temperature overdwinter storage. During the growing season, how- ever, high temperature speeds up the bulbing response. Flower formation was, for a long time, believed to be the result of short-day photoperiods, but Heath and Holdsworth show that, at the low temperatures necessary to inhibit bulbing, flowering is actually stimulated by long-day conditions. They postulate a rather complica- ted balance between a bulbing hormone, a flowering 27 hormone and auxin; together with their precursors and alternative, inactive end-products (see THEORIES). Mur- neek (75, 78), as has been previously pointed out, suc- ceeded in partially bypassing the photoperiodic require- ment of Rudbeckia by a high temperature treatment, and this has also been reported with China aster (5). Borthwick and Parker (15) studied the erreot of localized low temperature on flower initiation in B1- loxi soybean, in which the petiole and growing point were cooled separately. They found that in cooling the petiole or the growing point to 10° C. there was only a relatively small effect on flower-bud formation, while their previous work (87) had shown that cooling the en- tire plant to 12.50 C. during the dark period, flower- bud formation could be almost entirely prevented. They concluded that the production of the flowering stimulus in the leaf was the temperature-sensitive reaction. In a further report (90), they confirmed this conclusion by cooling or heating the leaves while the rest of the plant remained at normal temperatures, demonstrating that the limits of photoperiodic induction in Biloxi soybean are narrower than the growth temperatures. Long (65) found that in cocklebur, lowering the temperature to 40° F. would increase the induction period from one day to about eight days, but it increased the critical period only slightly. The effects of temperature on the photOperiodic re- actions are still not well understood, but it seems quite 28 probable that the effects are multiple. Temperature ef- fects the rate of growth of plants and thus would influ- ence the time of attainment of the "ripe to flower" con- dition, and the rate of the growth response to the stim- ulus. It has also been shown (above) that temperature has a specific effect on the "dark reaction" in short- day plants, confining the reaction to a comparatively narrow range of temperatures; and, within this range, regulating the critical period and the induction period, probably through its effect on the rate of chemical reactions. It might be suggested that this effect on the critical period and the induction period could be the result of : (l) the quantity of the stimulus pro- duced in a singledark period, (2) the partial des- truction or dissipation of that already produced, or (3) an effect on the effective distribution of the stimulus produced. It would seem feasible to test these and other possibilities. If, as seems quite clear (see THEORIES), two separate reactions take place during the dark per-l iod, it would be of interest to determine which of the reactions is the temperature sensitive. The author has attempted to study this question, but without definite results to date. Action spectrum work (see LIGHT AND PERCEPTION) has produced evidence that the dark reactions necessary for flowering in short-day species may be similar in nature to a dark reaction in long-day plants which in- hibits flowering. The action spectra for the effective 29 interruption of the dark periodshave been shown to be the same, thus it would seem logical to pr0pose that the photochemical reaction involved in both cases could effect, so strikingly, only reactions which are similar in nature. It would be of interest to determine wheth- er the effects of temperature on the dark reactions of the two types are comparable, as well as to investigate, by other means, the nature of the two reactions. The apparent demonstrations that some plants are long-day plants at some temperatures and indeterminate at other temperatures (105) might be explained by this phenomena. It might be prOposed that within a relatively narrow range of temperatures the dark reaction could take place (as has been shown to be true of short-day plants, see above) resulting in an inhibition of flowering, while outside this range the inhibition would be prevented. Vernalization, a prolonged chilling treatment re- quired by some plants before flowering will occur, has been recently reviewed by Whyte (123). The biennial Hypscyamms ni er, apparently a normal vernalization re- quiring plant, has been shown by Melchers (69) to flow- er in the first year if a scion of a short-day plant (maryland Mammoth tobacco) is grafted to it; and the plants are kept on a short-day. He preposes a second hormone "vernalin" normally present in annual plants but requiring a cold treatment for activation in bien- nial species. These results do not contradict Hamner's hypothesis (see THEORIES) and may be considered to lend 30 some support to it, although Hamner and Bonner (48) have shown that the dark "condition" in Xanthium is not transmissable. Stout (115) has shown that in beets, an annual beet grafted to a biennial will induce the bien- nial to flower, supporting the idea that the biennial is incapable of production of the flowering stimulus before cold treatment; and that the plant is capable of responding normally to the stimulus. It has been shown (29) that the effect of the cold treatment can be localized in the growing point, which suggested that the effect was a modification of the po- tentialities of the embryonic region, but the more re- cent evidence (above) suggests that the effect is on the young leaves. Gregory and Purvis (42) have shown that, in winter wheat and rye, vernalisation may take place in the seed of the subject plants before it has ripened, and they found that the effect was directly on the embryo, but it would not occur in vaccuo. It is not clear whether the cold treatments required by the grasses is comparable to the treatments required by the biennial dicotyledonous plants. 51 EFFECT OF OTHER ENVIRONMENTAL FACTORS Considerable early work attempted to relate photo- period to the carbohydrate: nitrogen ratio (2, 83, 88), but no definite correlations have been found. In prob? ably the most intensive work, Murneek (76, 79) found that upon induction, soybeans increased in nitrogen with respect to any form of carbohydrates. It has been abundantly shown that nutrients can markedly affect the number of flowers as well as the subsequent fruit-set (58, 81); but nutrition has never been proven to sub- stantially affect the critical period or the induction period of photOperiodically sensitive plants. As the experimental work in this paper apparently shows (see RESULTS), water-tension, or the effect of water tension on nutrition, seems to effect the rate and quantity of flower production but it has not been shown that it has any qualitative effect. 32 THEORIES ON THE MECHANISM OF PHOTOPERIODISM The theories of the mechanism of photOperiOdism represent, to date, attempts to formulate, from the a- vailable data, the step—wise reactions taking place within the plant which result in the production of the flowering stimulus. The first real effort was that of Hamner (44) who, it appears, directed his experiments to that end. His hypothesis represents the best one to date, in the opinion of the author. Hamner originally preposed a general scheme to as- sist in explaining the reactions occuring in cocklebur and Biloxi soybean. More recently he has expanded the concept to include all types of photoperiodically sensi- tive plants (47). This expanded hypothesis has not yet been published in full, but Snyder (115) has presented that portion which is applicable to long-day plants. In his hypothesis for short-day plants, Hamner has prOposed to let the symbol "A" represent the result of the re- actions taking place in the light period, and "B" the result of those taking place in darkness; then "G" would represent the summation of "A" and “B”, the substance or condition which moves to the growing point and there induces the flowering response. By exposing cocklebur plants to cycles of three hours darkness followed.by three minutes light for twenty-four hours or longer Ham- ner showed that flowering would not occur if this treat- ment was immediately followed'by the usual sixteen hour 55 dark period. It was apparent then, that some precursor of the stimulus, normally produced in the light period and utilized in the subsequent dark period, had been dissipated by the short cycles. This was confirmed when it was shown that, when the plants were given a relative- ly short, bright light period after the series of short cycles and before the long dark period, the plant would flower; and that the intensity of flowering was prepor- tional to the length and intensity of the light given, up to a maximum. These experiments showed that "A" was produced in the light, and was an essential precursor, which must be present at the beginning of the dark per- iod, in order that the dark reaction could occur. It appeared that "A" might be some product of photosynthe- sis, and this idea was given support when Borthwick and Parker (89) showed that, in Biloxi soybean, induction was directly preportional to photosynthesis. More re- cently Bonner has found, in unpublished experiments, that in the cocklebur the initial light period may be replaced by the infiltration of sugar or citrate into the leaf (7). In his early work (above) Hamner also found that if the initial light period.was followed by a long exposure to very low intensity light, the plant would fail to flower. He assumed that "A" would gradu- ally disappear under these conditions. ‘ ‘ The substance or condition "B" is preposed to be the result of the dark reaction,-and to act in a more or less catalytic manner after it has reached a 34 threshold value (the critical period), resulting in a transformation of "A" into ”C", the flowering stimulus. The "B” is preposed to be the-light sensitive portion of the reaction, being negated by as little as one min- ute of low intensity light in the middle of the dark period. The "C" is the final product of the reactions oc- curing in the leaf; it is the stimulus which moves to the growing point and there induces flowering. In his more recent extension of the hypothesis to include other types of photoperiodic plants, Hamner uses the same symbols to represent the same reactions or con- ditions. In long-day plants he assumes that the "B” is light stable and always present, so that the limit- ing factor is the amount of "A", the product of photo- synthesis, or the interaction resulting in the produc- tion of the stimulus, "Cm. Snyder, as mentioned above, has reported that plantain, a long-day plant, using light and dark cycles of other than twenty-four hours, will flower in long light periods regardless of the length of the dark period, but that with short light periods it will flower only if the accompanying dark periods are also short. This supports the hypothesis, for, if in short light periods something is produced which disappears when the succeeding dark period is too long ("A“); while in long light periods a stable product is formed (“C"), as the above experiment suggests, then this follows the prOposed hypothesis. The only 35 unexplained point would seem to be the disappearance of the “A" during the dark periods. It has been shown that the action spectra for both long- and short-day are iden- tical. This must mean that the disappearance of the "A" in long-day plants is the result of a positive reaction, presumably the same reaction which results in the for- mation of “B" in the short-day types. This paradox has not as yet been clarified, although it is possible that the level of auxin in the plant may also play a role here (see below). Hamner has also proposed that the hypothesis might also apply to those plants which require vernalization before they are capable of flowering. He suggests that in this case the formation of “B" may be dependent on the cold treatment, after Which it is stable as in long- day plants. The plants would then react as long-day or daybneutral plants. ‘It was pointed out earlier that Hamner has observed that, of those plants which require vernalization, same may subsequently behave as typical long-day plants and others as indeterminate, but none, so far as is known, show the characteristics of short- day plants. In further support of this idea Gregory and Purvis (42) have shown that in winter rye and wheat the cold treatment can be replaced by a treatment with short- day in rendering the plant capable of subsequent flow- ering, but these plants still required a subsequent long- day treatment before flowering would occur. One point, concerning both long-day and vernalization requiringw 56 plants which has not as yet been clarified is that if "A" is a typical product of photosynthesis, and it is limiting in long-day plants, then sugar-feeding should reduce the critical light requirement. This has been shown to be true only in Hyosgyamus, an apparently aty- pical long-day plant (65). It may be that in most long- day plants both sugar and auxin are deficient; this point should be investigated. Gregory (41) has attempted to show that Hamner's scheme for short-day plants is untenable in its present form, but he begins by misinterpreting the scheme. He assumes that as "B" is formed during the dark period it immediately reacts with "A" to form "C". He goes on to point out that, if the scheme is as he interprets it, certain difficulties arise. If ”B", the light sensi- tive reaction, reacts with "A“ as fast as it is formed, then the sensitivity cannot be accounted for. If, on the other hand, the "B" accumulates during the dark per- iod then the quantity or "A" would limit the rate of the reaction, and this is not true. Gregory goes on to propose that the "A" is rever- sibly converted to "B", and the "B" is then moved out of the leaf during the dark period. At the critical period the quantity of "B" translocated out of the leaf is supposed to have reached the critical value required to induce flowering. However Hamner (48) has shown that it takes four days for the stimulus to move out of the leaf, and that this is apparently not a function of the 37 quantity of the stimulus. Under the hypothesis as pro- posed by Hamner, Gregory's objections are no longer val- id Bunning (16, 17) preposes to explain the photOper- iodic phenomena through a diurnal rhythm which.he as- sumes to be present in all plants, and for which.he has considerable experimental evidence. This rhythm is characterized by two phases which he has named the pho- tephile phase and the skotophile phase. They represent quantitative and qualitative changes in the biochemis- try of the plant. The photOphile phase, as the name implies, is the light phase, characterized by a stimu- lation of flowering when light is applied; while light applied during the skotOphile phase tends to inhibit flowering, especially in short-day plants. Light acts to start the rhythmic cycle and when further light is applied during the ensuing photOphile phase it reinfor- ces the momentum.of the cycle and promotes flowering. In long-day plants, because the cycle does not start un- til several hours after the initial light stimulus, a small amount of light, offered after several hours of darkness, will be introduced at the time of the maximum of the photOphile phase and thus promote flowering. If the light is offered later (after 12-15 hours in long- day plants) it will arrive at the time of the skotOphile phase and the plant will not flower. The light effect in long-day plants is a reinforcement of the photOphile phase rather than an inhibition of the skotOphile, as‘ 58 shown by the fact that long-day plants flower readily in continuous light. It would appear from this that when a plant is on one rhythm, the experimental dark period would have to coincide with the existing skotophile phase in order to be effective in promotion or inhibition of flowering (depending on the type of plant). For long-day plants the critical experiments have not been performed, while in the case of shortfiday types the existing evidence is conflicting. Cocklebur may be removed from the long- day cycle at any time during the light period (after a brief minimum, see above) and the critical period is found to be the same for the one day induction. Biloxi soybean, however, offers some support for the hypothe- sis, in that it has been shown that this plant has an optimum light requirement previous to the dark period, but this could also be explained by a gradual dissipa- tion of the product of the light reaction, as has been shown to occur under low intensities of light (44). It may also be suggested that the stimulus which.was pro- duced during the last cycle was adversely affected by the continued light. On the basis of the present evi- dence this theory cannot be rejected but it seems rather fruitless as a working hypothesis. It suggests no new approaches to the ultimate problem, the biochemical re- actions which result in flowering; instead it would seem to set us back another step: to the study of the rhy- thms which.make possible the reactions which produce 59 the flowering stimulus. While this would be an inter- esting and worthwhile problem, its solution would not be necessary in order to carry on the present work, and in fact, an elucidation of the reactions resulting in the production of the flowering stimulus might be of great value in the study of the pr0posed cycles. It should be kept in mind, however, that when the problem reaches the stage of identifying the basis for the spe- cific reactions involved this idea may play a very im- portant part. Lang and Melchers (62) prOpose a general scheme to outline the reactions taking place in Hyoscyamus gig@£_ which results in either flower formation or further ve- getative growth. They pr0pose two separate reactions, one (the primary reaction) taking place independent of light, is that which directly promotes flower formation, after it has attained a critical threshold level. The second reaction (the secondary reaction) takes place on- ly in darkness, and it acts to inhibit the primary, flow ering reaction. The products of the primary reaction, presumably the "flowering hormone", may accumulate, thus producing the induction phenomena. In this form the hypothesis fails to take into ac- count the observed flowering by other long-day plants on short cycles of light and darkness (57) but it appar- ently holds true for Hyoscyamus. It should be pointed out that some of the other reactions of Hypscyamus are apparently unique. Defoliation and sugar-infiltration 40 of the leaves will cause flowering in Hyoscyamus (61) but this does not seem to be true of other long-day species. Cholodny (26) in 1959, suggested that the experi- mental evidence at that time did not preclude the possi- bility that auxin was the controlling mechanism of flow- ering. Experiments with auxin (Indol-Shacetic acid) have since shown that while it is not the flowering hor- mone, it may play an important part in the production of the hormone (10). Recent experiments have shown that when cocklebur is kept very near the critical period, the application of 2,5,5 Triiodobenzoic acid, an "anti- auxin", will result in the initiation of flower-like forms at the growing point (6). This seems to be fur- ther indication that the dark reaction in short-day plants may be, in part at least, a reduction of the aux- in concentration at the site of production of the stimu- lus. Unpublished experiments by the author suggest that applied auxin stimulates and accelerates stem.elon- gation in certain long-day plants (especially Raphanus sativus), but the effect on flowering itself has not been determined. LeOpold and Thimann (65), in investi- gating the effects of applied auxin on the formation of flower primordia in long-day and short-day plants, found that applied auxin at any concentration would inhibit flowering in short-day plants, but in the case of the long-day species low concentrations of auxin increased the number of flower primordia markedly. This increase 41 in flower primordia was directly correlated with the growth.rate of the plant, as measured by weight. At higher concentrations of auxin the production of flower primordia was suppressed also in the long-day type; and in no case was flowering induced when the plant was not on an inductive cycle. These results may be fitted in- to the hypothesis of Hamner, if it is assumed that in long-day plants auxin is limiting the production of the stimulus, while in the short-day types the concentration of auxin is greater than the Optimum and is inhibitory. The effect of the long-dark period in both types may represent a reduction in active auxin, in the later case to a level which permits the production of the stimulus and in the former causing an even greater de- ficiency. This idea has not yet been critically tested, but it would seem feasible to do so. Heath and Holdsworth (50) as already mentioned, (see TEMPERATURE) have done a great deal of work with the onion and its peculiar reactions to various environmen- tal stimuli. They have found that bulbing is the re- sult of a longeday stimulus in the presence of high.tem- peratures. The stimulus was found to be perceived by the youngest emerged leaf blade, and it was irreversible after an adequate induction period. They propose a novel explanation for the critical period required to induce bulbing, but unfortunately it is apparently not applicable to other long-day pheno- mena. They pr0pose that under the influence of "B" 42 (their bulbing hormone) the linkages of the molecules or micelles of the cellulose making up the cell walls are broken and, given sufficient time, new cellulose is deposited between the old. If the length of the light period is insufficient, the "B" is not longer pres- ent to deposit the new cellulose, and the cell wall re- turns to its original dimensions. The "B" is produced from a precursor "A" which is formed in light. The "A" is transformed to "B" as it is formed, under high tee; peratures, in a reversible reaction; at lower tempera- tures the "A" is irreversibly changed to "C", considered a leaf-growth hormone. Experimental evidence has shown that the critical period is shorter at high temperatures and also decreased with age. They explain this by pro- posing that the higher the temperature, the more "A" goes to “B" instead of to "C". In older plants the greater leaf surface results in the production of more "B" during the light period which persists longer into the succeeding dark period and the greater quantity of "B” should result in more rapid deposition of new cellu- iose. Heath and Holdsworth have found that the induction period for bulbing of the onion is independent of no- ticable swelling, which seems rather difficult to recon- cile with.the above hormonal mechanism. They suggest, however, that the induction period may be associated with the failure of the plant to produce new roots, al- though they admit that this has not yet been subjected 43 to experimentation. The preposed substance "C" is produced from."A", in mature leaf bases, apparently quite independent of the temperature, and it may also be formed in the grow- ing point in storage, but this reaction takes place only at high temperatures. "C" is proposed to be essential for flowering, by inducing auxin formation in the elon- gating scape. To account for the flowering response of the onion, Heath and Holdsworth pr0pose another hormone system, the two forms of which they call "E" and "F". These two related hormones are considered to be in equilibri- um, and both are destroyed by high temperature. They suggest that either "E" or "F" can promote inflores- cence initiation but "3" is necessary to induce floral initiation and hence scape elongation. By removing the swollen leaf bases before storage in the fall or in the spring and comparing the flowering response, they have been able to show that the flowering stimulus is pres- ent in these swollen bases in the fall and gradually diffuses into the growing point during the storage per- iod. During the winter, according to their hypothesis, the "E" is gradually changed to "F". Sometime in the spring the threshold value of "Fh is reached to permit flower initiation, the length of time required depend- ing on the size of the set. The "F" initiates the in- florescence toward spring; then, when growth resumes, the now active root system permits the change of "F" 44 back to "E", which induces scape elongation. As both of these hormones are destroyed by high temperatures, flowering is prevented by a very warm spring or delayed planting until the weather is warm. Of-the schemes which have been discussed here, the only one which seems applicable to an entire group of ‘ plants is that of Hamner. There appears to be no valid experimental evidence which would render this scheme untenable in any short-day plant. This may be due, in part, to its apparent simplicity, yet it represents a significant advance, in that it provides a basis for fur- ther elucidation of the mechanism of the photOperiodic response, and experiments to this end are now in prog- ress. With regard to the extension of Hamner's hypoth- esis to include long-day and vernalization requiring plants the experimental evidence is not so clear, as has already been pointed out; and further work will be required to settle this question. III METHODS AND MATERIALS The experiments described were performed in the horticultural greenhouses at Michigan State College be- tween March and September of 1949. The greenhouses were of the conventional type, steam heated when the temperature was low and ventilated when it became too warm. As these experiments were conducted from early spring and continued throughout the summer a wide range of temperatures were encountered, ranging from the low- est night temperatures of 50° F. to an occasional high of 1050 F. The consistency of the experimental results and the uniform.behavior of the controls provide strong evidence that temperature within the extremes cited has little or no qualitative effect on the flowering re- sponse of these plants. To regulate the photOperiod two frames were con- structed over which double thicknesses of black cloth could be drawn (Fig. 2). One was normally closed at four P.M. and Opened at eight the following morning. The other was equipped with two pairs of white flores- cent lights attached through a General Electric time clock so that the photoperiod in this frame could be regulated. For the determination of the critical period these lights were regulated to turn on at four A.m. and go off after eight A.M., and to be on again from four P.M. to eight P.M. In later experiments they were on from sunset 46 to midnight and from three A.M. until sunrise, thus giv- ing an adequate long-day. Near the experimental set-up another florescent light was installed and kept burning throughout the night to provide a twenty-four hour photOperiod when required. Seeds of Gypsophila eleggns, var. New York Garden Market, were obtained from commercial sources; and the cocklebur fruit was kindly provided by Dr. Beale of the University of Chicago. 47 EXPERIMENT 1, A Study of the Critical Period and the Induction Period in Gypsophila Elegans. Preliminary observations indicated that gypsOphila ele ans, a long-day plant, was sensitive to extremely weak intensities of light supplementing the day light of the winter months. The first experiment was designed to find the ap- proximate critical period and to investigate the extent to which induction would take place in this plant. Two flats of GypsOphila elegans were sown on March twenty-second and immediately placed on an eight hour day in the greenhouse. On April tenth, uniform.sized seedlings were selected and transplanted to one hundred twenty 4%" unglazed clay pots in good greenhouse soil, three plants per pot. On May sixth, after the seedlings had resumed vig- orous growth, they were placed under the experimental light periods of eight, ten, twelve, fourteen, sixteen, and twenty-four hours, six pots in each period. An ex- tra twenty-four pots were moved to the sixteen hour per- iod and at the end of two days, six of these pots were moved back to the eight hour photOperiod. At the end of five, ten, and twenty days other groups of six pots were returned from the long-day to the short-day bench. The remaining six pots were left on the sixteen hour day for the duration of the experiments. In order to expose the plants to the varying day t lengths, the pots for the ten, twelve, and fourteen hour days were set in wood flats to facilitate handling, and were moved from one frame to the other according to the following schedule: During the day: 10 and 12 hour pots moved to the ' long-day bench At 6 P.M.: 10 and 14 hour pots moved to the short-day bench After 8 P.M.: 12 and 14 hour pots moved to the long-day bench Thus all plants were exposed to eight hours of sun- light and varying periods of artificial illumination each day. It may be assumed that the quantity of photo- synthesis taking place in all plants was essentially e- qual. The results were recorded on June twenty-first when most of the plants on long-day were flowering profusely. Measurements were made from soil level to the tip of the stem or to the uppermost flowers, when these were pres? ent. The plants were continued on the various day lengths for another two weeks and no qualitative varia- tions from these results were observed; and, in addition, the eight hour controls were allowed to remain for an- other six weeks. 49 EXPERIMENT 2, Attempts to Transfer the Flow- ering Stimulus in GypsOphila Elegans. At the same time that experiment 1 was run, a sec- ond experiment was attempted to discover if Gypsophila elegans could be induced to flower by the injection of extracts of flowering plants. The general method of Grainger (40) was used. Plants beginning to elongate were selected for extrac- tion, cut into small pieces and ground in a mortar. An amount of distilled water was added to approximately double the volume and the mixture was filtered through a coarse cloth. The mixture was injected by means of a hypodermic needle and syringe into several of the leaves and petioles of each of the ten test plants. It was es- timated that approximately 1.0 c.c. was injected into each plant at each application. Sixteen applications were made beginning on May twenty-fourth and continuing until June eighteenth, cov- ering a period of twenty-five days.. On June eighteenth the injections were discontinued and the plants remained on an eight hour day for the duration of the experiment, a period in excess of two months. It should be noted that the injections, while not administered daily, covered a period exceeding the time of visible response of plants put on a long-day. 50 EXPERIMENT 5, Attempts to Transfer the Flowering Stimulus in Xanthium Pennsylvanicum. This experiment was conducted to combine the appli- cation of 2, 5, 5 triiodobenzoic acid, shown by Bonner (6) to effect a "partial initiation of the inflores- cence" in Xanthium, with the here-to-fore unsuccessful injection technique in an attempt to induce floral ini- tiation in cocklebur. One hundred Xanthium fruit were soaked twenty-four hours in tap water and planted one in each 2" pot on July eighth. They were immediately placed on a long- day (twenty-one hours). On July twenty-eighth they were transplanted to 5%" pots and on August second, after they had recovered from the shock of transplanting, treatments were started (Table 2). The short-day treat- ‘ment was provided by placing the plants on the regular short-day bench with eight hours of light and a sixteen hour dark period. Extracts were made from complete Xanthium.plants which had been placed on short-day and were showing mac- roscopic flower formation, in the same manner as des- cribed in experiment 2. The extracts were injected in- to the petioles of 2-5 of the youngest fully expanded leaves of each test plant. Again a total of approxi- mately 1.0 c.c. of extract was injected into each test plant. The treatments were continued every day for six 51 days, at which time they were discontinued and all plants were returned to the long-day bench. The results were recorded fourteen days later, at which time the plants were dissected and examined as described by Hamner and Bonner (48). The results appear in Table 2. There was no effect by the treatments. 52 IV RESULTS It has been shown (Table I) that, under the condi- tions employed for these experiments, the average criti- cal period.for gypsOphila elegans is approximately elev— en hours. Those plants whose stems were less than one centimeter long were still rosettes, and had shown no visible indication of future flowering. As has been previously mentioned, those pots which were exposed to the ten, twelve, and fourteen hour pho- taperiods were set in flats to facilitate transfer; but those pots kept on the eight, sixteen, and twenty-four hour photoperiods were set in sand and were not dis- turbed during the course of the experiment. It was noted during the course of the experiment that the four: teen hour pots showed the first visible response, fol- lowed by the twenty-four, sixteen, and twelve hour pots respectively. It can be seen that this order follows that of the ultimate degree of response. If it is as- sumed that, on daylongths greater than the critical, the degree of response is correlated with the length of day; and that a variation in water-tension and mechanical disturbances accelerate the rate of response, then the observed results can be quantitatively accounted for (Figure 1). That the response is related to the length of day is shown for those plants in which the other treatments are comparable, so that the only assumption which need be made is that mechanical disturbances ac- 53 celerate the rate Of response in this plant; as van Overbeek (86) has shown to be true with the pineapple. In the second part of this experiment, where an attempt was made to determine whether induction could be observed in this plant, it was found that nO induc- tion would occur. NO elongation was apparent in any of the induction series, except for those plants which had received a long-day treatment for twenty days. Five Of these plants showed some elongation (up tO eight. centimeters) before being transferred back tO the short- day. Upon transfer to the short-day the elongation im- mediately ceased. On two Of the plants which had flow- er-buds at the time Of the transfer, the flowers Opened and appeared normal but they were without pedicels (Fig- ure 5). 4— Some Of the plants Of gypsOphila elegans were al- lowed to remain on an eight hour day for a total of five months. By the end of this time many Of these plants had died while others showed the develOpment Of one or many lateral buds, producing "secondary rosettes". Throughout the entire course Of the work no plant was ever Observed to begin stem elongation or flowering while on the short-day. The injection experiments showed no promise Of 81106838 0 Table 1. Length Of stems Of gypsophila elegans exposed to varying daylengths for forty-five days, as an indication of the flowering response. Hours Of Light per Day 8 10 12 14 16 24 0.5 0.5 0.8 0.5 1.0 3.0 0.5 0.5 0.8 14.0 1.0 12.0 0.5 0.5 2.5 15.0 10.0 14.0 0.5 0.5 1.0 17.0 0.5 1.0 0.5 0.5 7.0 24.0 1.5 8.0 0.5 0.5 8.0 11.0 3.0 17.0 0.5 0.5 0.5 14.0 0.5 8.0 0.5 0.5 2.0 15.0 0.5 9.0 0.5 0.5 14.0 3.0 12.0 14.0 0.5 0.5 0.5 15.0 0.5 15.0 0.5 0.5 1.0 20.0 0.8 18.0 0.5 0.5 1.0 15.0 9.5 19.0 0.5 0.5 17.0 17. 0.5 2.0 0.5 0.5 4.0 24.0 2.0 10.0 0.5 0.5 14.0 14.0 2.0 18.0 0.5 0.5 0.8 15.0 0.5 14.0 0.5 0.5 0.8 15.0 0.5 17.0 0.5 0.5 4.0 (dead) 1.0 17.0 0.5 1.5 4.0 0.5 14.0 18.0 1.0 15.0 20.0 005 005 404*103 1406*104 407*104 lZeOile“ Average Length Of Stems (in centimeters) {snug - Léfifl * S.D.= . . n(n-I) 55 Table 2. The effect Of plant extract injections and 2,3,5 triiOdObeninc acid on the flowering response Of Xanthium pennszlvanicum. Control f f f f f f f f* Water Control f f f f f f f f SHORTinflY' Extract Injection f f f f f f f f "' TIBA rrrrrrrr 'In'iectionaTIBA r rrrrrr 1' Control v v v v v v v v Water Control v v v v v v v v LONG-DAY’ Extract Injection v v v v v v v v 4 TIBA v v v v v v v v Injection &,TIBA v v v v v v v v _-_ s f - flowering; v - vegetative 56 W\. H 0 plants mechanically 1; stimulated a o +:<3_ '§.H '2 o 3, a o 4.3 on plants '8 not 'G K\ stimulated s’ . g ,. .4 x” ’// //’ 1,! ,/’ / l,r / L o” <3 in i i . . 8 lo 12 14 16 24 7 Hours of Light per Day Figure 1. The relationship or the length or the light period and mechanical stimulation to the flower- ing response (as indicated by stem elongation) in.§yp- saphila slogans. 57 Figure 2. Experimental bench.used for regulating the photoperiod. Figure 5. Typical plants from each Of the six photoperiods. Left to right: 8, 10, 12, 14, 16, 24 hours of light per day, after forty-five days. Figure 4. Comparison Of plants on the fourteen hour photOperiOd (left) with those on the sixteen hour photoperiod (right), forty-five days after treatments were started. Figure 5. Gypsgphila elegans which.had previously been exposed to a twenty day induction period, after fortybfive days. 59 V DISCUSSION AND CONCLUSION A number Of plants extremely sensitive to photOper- iodic stimuli have been described and studied in the past few years. The study Of these plants has furnished most of the present knowledge Of photOperiOdism. The discovery Of other plants also sensitive could conceiv- ably result in a significant increase in the knowledge of photOperiOdism. One of the most significant advances that could be made toward the isolation and identification Of the flowering hormone would be the successful extraction Of an active principle from an induced plant, which upon application to a vegetative plant would induce flower- ing. It may be found that the plants in which photoper- iodic induction is the most pronounced are not necessar- ily those which may prove most successful for extrac- tions. In fact thousands of extraction and injection attempts have been made (9) especially with cocklebur and other very sensitive photOperiOdic plants, but no successful results have ever been substantiated. In the attempts Of Bonner and Hamner (9) a few apparently successful results were Observed but they could not be consistantly reproduced. Several reports have been made Of varying degrees Of success using plant extracts (4, 8, 69, 105) but all are without confirmation. However, it would seem that further attempts to 60 extract the active principle are not likely tO prove fruitful until more can be discovered regarding the na- ture Of the stimulus through other techniques, or unless some new combination of known facts can give specific suggestions for experimental techniques. For example, Bonner (7) points out the essential similarity between the nature Of the stimulus, so far as it is known, and the nature Of certain of the graft- transmissable viruses. Should this comparison prove to be valid it would suggest the great difficulties in- volved in any attempted extraction and re-introduction. Galston (53) points out the possible reasons for failure: (1) unsuccessful extraction, (2) inadequate concentration, (5) unsuccessful injection, or (4) lack of suitable bioassay. This assumes that the active principle can exist independent Of living cells, an as- sumption which remains, at the moment, Open tO question. In the experiments described herein it was, of ne- cessity, tentatively assumed that by its nature extrac- tion and reinjection is possible. Attempts were made to take into consideration the four factors listed above in various ways as described. The experiments were two fold in their aim. In ex- periment 2 typical injections were carried out in an effort to discover a plant which.might react where num- erous others had failed; while in experiment 5 reference was made to the recent work of Zimmerman and Hitchcock (128) and Galston (52) in which they found 2, 5, 5, 61 triiOdObeninc acid when applied to plants acted as an inhibitor Of auxin activity and that Of Bonner (56) where he applied TIBA to cocklebur held very near the critical period and Observed the formation of the in- florescence-like primordia at the apex, but no flowers. This suggested that perhaps the partial stimulation of the TIBA could be combined with an extraction and in- jection technique to induce flowering, and this was at- tempted. Since these experiments were performed, Bonner (7) has apparently induced flowering in cocklebur with 2, 5, 5 triiodobenzoic acid when the plants were exposed to conditions very close to the critical period by giving an adequate dark period but interrupting it in the mid- dle with a "spot" Of light. This would seem further in- dication that one Of the reactions taking place in the dark period, apparently the one most light sensitive,’ is the lowering of the auxin level. 2. 5. 6. 62 VI SUMMARY The literature relating to photOperiOdism is dis- cussed from the point Of view Of possible explan- ations Of the photOperiOdic phenomena. The critical period Of Gypsqphila elegans was de- termined and found to be between ten and twelve hours Of light. Environmental factors other than photoperiod were found to affect the rate of response Of gypsgphila elegans to photOperiod. gypsOphila elegans was found to be incapable Of in- duction. Attempts were made to induce flowering in CypsOphila elegans by injecting the brei Of flowering specimens into vegetative plants Of the same species, without success. Attempts were made to induce flowering in Xanthiwm pennsylvanicum by injecting brei made Of flowering specimens into vegetative plants of the same spe- cies and in addition treating a portion Of the in- jected plants with 2, 5, 5 Triiodobenzoic acid, with- out success. 1. 2. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 63 VII LITERATURE CITED Allard, H.A. An interesting reference to length Of day as affecting plants. Science 99: 265, 1944. Auchter, E.C. and Harley, C.P. Effects Of various lengths Of day on develOpment and chemical com- position Of some horticultural plants. Amer. Soc. Hort. Sci. Proc. 199-202, 1924. Bailey, L.H. Greenhouse notes for 1892-95. I. Third report on electrO-culture. Cornell N. Y. Agr. Exp. Sta. Bull. 55: 147-157, 1895 Behrens, Gertrud. 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