A comparison of the effects of colchicine and some purified vera, trum alkaloids on nuclear division in roots of allium cepa: L; with preliminary observations on germination root length and environmental factors

This study represents an investigation and comparison of the effects of various Veratrum alkaloids on nuclear mitotic division in an effort to determine 1. whether some purified alkaloidal mixtures or crystalline alkaloids possess the ability to modify the cycle of nuclear and cellular division, and 2. to determine how such activity compares in kind a degree to that already well established for colchicine. In addition, preliminary studies have be conducted to determine what effect a number of physical and chemical factors have on nuclear mitotic division being studied in order to eliminate their influences as experimental variables. Onion bulbs, Allium cepa var. White Portugal, were used as the experimental plant. Roots were sprouted on the bulbs using tap water and experiments were conducted under constant temperature (30°C.±0.5°C.) conditions. Roots from 1.5 to 6.0 cm. in length were used for all observations of nuclear division. Microscopic examination of the cells was performed by root tip smear method in which root tips were killed and fixed for 24 hrs. or longer, softened to separate the individual cells, rehardened, stained, and observed through a microscope at magnification of 440 diameters. All intact cells observed in a predetermined area of a glass slide, either resting or in any stage of mitotic nuclear division were recorded and the per cent of cell in the process of nuclear division at the time of observation was calculated. Suitable statistical technics were employed (chi-square, linear regression, and analysis of variance) to analyze the experimental results and permit conclusions to be drawn there from. No significant differences were noted from control values in nuclear division between times of exposure of 3 and 6 hrs. to a 2 per cent solution of 2-chloreothanol. Three hr. bulb soaking failed to stimulate germination of a significantly greater number of bulbs than was produce by tap water alone; however, production of new roots was significantly increased over results observed for tap-water controls. These data suggest that pre-soaking periods for Allium bulbs are not harmful to nuclear mitotic division. Further, the agent appears to be incapable of breaking bulb dormancy in the concentrations used and for soaking time employed by is highly effective in increasing the germination rate of new roots from non-dormant bulbs (approximately 19 per cent over control values). Although there was no significant difference in over-all nuclear mitotic division between roots up to 7.5 cm. in length, very short roots (up to 1.0 cm in length) were consistently low (2.4 per cent) in the number of dividing nuclei from that of a control (4.0 per cent) which suggests that short Allium roots up to 1. cm. long increase in length more from cell elongation than by meristematic mitotic production of new cells. To eliminate the influences of root length on nuclear division as an experimental variable, only roots from 1.5 to 6.0 cm. long were used for all observations. In a population sample of 40 Allium bulbs the number of mitotic dividing nuclei was not significantly changed for different times of day when observations were performed on roots obtained every 3 hrs. over a 24 hr. period of time. The effects of light, intermittent light, and darkness on nuclear division and root length over a 72 hr. test period indicated that prolonged exposures to artificial light had a noticeable but not-significant inhibitory effect (P>.05) on mitotic nuclear division and a marked inhibitory effect on root length (P<.001). In order to eliminate the influences of light as an experimental variable on the number of mitotic dividing nuclei, Allium roots should be exposed to known intensities of light which may be controlled at all times. Exposure of Allium roots to 7 different temperature gradients from 15 to 45°C. in increments of 5°C. for 24 hrs. revealed that the mean number of mitotic dividing nuclei exposed to 20, 25, 30, and 35°C. were not significantly different from the mean values obtained from roots exposed to the control temperature (30°C.), whereas means at temperature extremes of 15, 40, and 45°C. differed significantly (40 and 45°C., P<.01) from controls. This suggests that rather wide temperature fluctuations may be permitted without producing undesirable alterations in mitotic nuclear activity. Temperature extremes, however, may have marked effects on mitotic processes; therefore, whenever possible, experiment should be conducted under well controlled temperature conditions. Examination of Allium bulbs treated with concentrations of colchicine of 0.1, 0.2, 0.4, or 0.8 percent for predetermined periods of time of 3, 6, 9, or 12 hrs. revealed that the time and concentration factors both caused significant changes in nuclear mitotic division from control values. Independent of the time factor, all colchicine concentrations had an inhibitory effect. When time was examined as the major variable, significant differences in nuclear mitotic activity were also observed. The data also showed the existence of a significant interrelation between the effects produced by the time and concentration factors. Typical colchicine-mitotic (c-mitotic) effects were produced with all colchicine concentrations had an inhibitory effect. When time was examined as the major variable, significant differences in nuclear mitotic activity were also observed. The data also showed the existence of significant interrelation between the effects produced by the time and concentrations factors. Typical colchicine-mitotic (c-mitotic) effects were produced with all colchicine concentrations employed. The data suggests that colchicine, studied by these experimental methods, is capable of modifying nuclear mitotic activity by producing typical c-mitotic changes in the chromosomes of Allium roots. Successful inhibition of the spindle apparatus may be achieved and polyploidy cell (2n = 32) are prominent. When using colchicine experimentally, not only must be concentrations be taken into account but also the exposure time must be considered. The effects produced by Veriloid, a standardized mixture of hypotensively active alkaloids present in Veratrum viride Ait. Were examined in Allium bulbs treated with acidified solutions which contained concentrations equivalent to 0.1, 0.2., 0.4., or 0.8 per cent of Reference Standard Alkavervir (Riker Laboratories in frees tape water for predetermined periods of time (3, 6, 9, or 12 hrs.). The data suggests that alkaloidal agents present in Veratrum viride have and effect on nuclear mitotic division in roots of Allium cepa L. Chromosomal aberrations were frequently observed although their appearance suggested a markedly different mode of action than that observed with colchicine. Polyploid cells may be noted and the effects on nuclear mitotic division are in agreement with those reported by Witkus and Berger (1944) for veratrine, an alkaloidal mixture obtained from a different plant source (Schoenocaulon officinale A. Gray) but whose constituents are structurally similar to those found in Veriloid. The effects produced by protoveratrine A, a purified crystalline alkaloid present in Veratrum album L. were examined in Allium bulbs treated with a 0.025 per cent acidified solution in fresh tap water for predetermined periods of time (3, 6, 9, or 12 hrs.). The results suggest that protoveratrine A is capable of modifying nuclear mitotic division to produce polyploid cells in roots of Allium cepa. The changes observe red were indistinguishable from the changes reported in the Veriloid study.

A COMPARISON OF THE EFFECTS OF COLCHICINE AND SOME PURIFIED VERATRUM ALKALOIDS ON NUCLEAR DIVISION IN ROOTS OF ALLIUM ~ L. with Preliminary Observations on Germination, Root Length, and Environmental Factors by Douglas Lee Smith A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacognosy College of Pharmacy University of Utah August, 1956 LIBRARY UNIVERSITY OF UTAH This Thesis for the Ph.D. degree by Douglas Lee Smith has been approved by er, Supervisory C ee /.~ ~- - - (~'t/f <:#7-:L:~) Rea~Supervlsory Committee Hg~ MaJor Department if !tI, I -ii- Appreciation is expressed to the several members of the staffs of the University of Utah College of Pharmacy, the Departments of Botany and Genetics of the University College, and the Department of Bacteriology of the College of Medicine~ To Dean L~ David Hiner, who supervised these studies and offered many helpful suggestions and criticisms; to Dr. Ewart A. Swinyard as Director of Pharmaceutical Research and to Dr. Irving B. McNulty whose interest and guidance provided much assistance; to Dr. Charles Mo Woolf for his contributions with the statistical procedures; to Dr. George E. Osborne who assisted in reading the thesis; and to Dr. Walter Po Cottam and Dro Louis P. Gebhardt for their interest as members of my committee, acknowledgment is gratefully made. Sincere gratitude goes to my family, especially my wife, Joyleen, who typed the thesis, for the constant encouragement and inspiration in this undertaking. I. II. III. - iii - TABLE OF CONTENTS GENERAL INTRODUCTION. • • · . . . • • • • • • • • GENERAL PROCEDURES. • • • • • • • • • • • · . • • THE EFFECTS OF PRELIMINARY TESTS ON NUCLEAR DIVISION AND ROOT GROWTH IN ALLIUM. • • • • • • • • • • • • • • • • • • • • • A. Effects of Chemical Treatment wi th 2-chloroethanol. • • • • • • • • • • 1. Introduction.... • • • • • • • 2. Methods •••••••••••••• 3. Results •••••••••••••• 4. Discussion. • • • • • • • • • • • B. Relation of Root Length to Nuclear Division. · • · • • · • • • • • • 1. Introduction · · • • • • • • • • • 2. Methods. • .. · • • • • • • • • • • 3. Results. • • • · · · • · • • • • • 4. Discussion · • · • • • • • • • • • Page 1 10 17 17 17 18 20 25 29 29 29 30 33 IV • THE EFFECTS OF ENVIRONMENT ON NUCLEAR DIVISION AND ROOT ELONGATION IN ALLIUM. • • • • • 35 A. Effects of Light. • • • • • • • • 35 B. 1. Introduction... • • • • • • • • 35 2. Methods. • • • • • • • • • • • • • 36 3. Results.. • • • • • • • • • • • • 39 4. Discussion. • •.• • • • • • • • • 43 Effects of Temperature on Nuclear Division. • · • · • I. Introduction • · · • 2. Methods. • • • • • • 3. Results. • • • • • • 4. Discussion • • • • • • · · · • • • · • • · • • • · • • • • • · • • • ·• ·· • · • • • • • 47 47 48 49 49 V. EFFECTS OF LILIACEOUS ALKALOIDS ON NUCLEAR DIVISION IN ALLIUM ROOTS. • • • • • • • • 52 A. Effects of Colchicine • • • • • • • • • • 52 1. Introduction.... • • • • • 52 2. Methods. • • • • • • • • • • • • • 54 3. Results. • • • • • • • • • • • • • 57 4. Discussion.. • • • • • • • • • • 66 VI. VII. VIII. - iv - TABLE OF CONTENTS (contfd.) Page B. Effects of Veriloid • • • • • • • 69 1. Introduction. • • • • • • • • •• 69 2. Methods. • • • • • • • • ••• 70 3. Results.. • • • • • • • • • • 71 4. Discussion. • • • • • • • 78 C. Effects of Protoveratrine A • • • • • • • 81 1. Introduction.... • • • • • 81 2. Methods 0 • • • • • • • • • • • •• 82 3. Results. • • • • • • • • • • • •• 82 4. Discussion. • • • • • • • • • •• 86 GENERAL DISCUSSION. I • • • • • • • • • • • • • • • 88 SUMMARY AND CONCLUSIONS • • • • • 94 . . • • • REFERENCES. ABSTRACT • • • 0 • • • • • • • • • • . . • • • • • • 100 Table 1 - v- LIST OF TABLES Estimates of Nuclear Division Means from 2=chloroethanol Effects • • e 0 " . o • .. • 2 Analysis of Variance of 2-chloroethanol Effects on Nuclear Division in Allium 3 4 5 6 7 Roots ••• e " 0 0 ......... . • • 0 • Estimates of Nuclear Division Means for Various Root Lengths • • • .. • .. o • • • Analys+s of Variance of Root Length Effects on Nuclear Division in Allium Roots ••••. · . · " Estimates of Nuclear Division Means for Various Root Lengths • • • • • • • • 0 • 0 • Analysis of Variance of Root Length Effects on Nuclear Division in Allium Roots ... " ... · . Estimates of Nuclear Division Means for Different Times of Day • • • • • .. • • • 0 • 8 Analysis of Variance of Time of Day 9 10 11 Effects on Nuclear Division in Allium Roots o D 0 0 0 0 0 0 0 0 • e •• 0 • 0 • Estimates of Nuclear Division Means for Light Changes and Darkness " • .. 0 • • • Analysis of Variance of the Effects of Changes in Exposure to Light on Nuclear Division in Allium Roots • .. • .". .. . " .. . .. · . • • • 0 Estimates of Root Length Means for Light Changes and Darkness • " • • .. . • 0 0 • • 12 Analysis of Variance of the Effects of Changes in Exposure to Light on Root Page 21 21 31 31 32 32 40 40 41 42 Length in All ium Roots • .. • • .. • • • • .. I. • • 42 13 Effects of Colchicine on Nuclear Division in Allium Roots (Main Groups--Concentrations) •• 59 Effects of Colchicine on Nuclear Division in Allium Roots (Main Groups--Time) •• 0 ...... 60 Table 15 - vi - LIST OF TABLES (cont1d.) Analysis of Variance of Colchicine Effects on Nuclear Division in Allium Roots. 0 •• Page 61 16 Effects of Veriloid on Nuclear Division 17 18 19 20 21 in Allium Roots (Main Groups--Concentrations) •• 72 Effects of Veriloid on Nuclear Division in Allium Roots (Main Groups--Ttme) ••• Analysis of Variance of Veriloid Effects on Nuclear Division in Allium Roots •• 0 Esttmates of Nuclear Division Means for Different Ttmes of Exposure to Proto-veratrine A. • • • • 0 .. 0 • • 0 .. • • • • • • • 41> 0 • • · . . . Analysis of Variance of protoveratrine A Effects on Nuclear Division in Allium Roots. . .. Summary of the Effects of Different Liliaceous Alkaloids on Nuclear Mitotic Division in Roots of Allium cepa • • • • · . . . 73 74 84 84 90 - vii - LIST OF FIGURES Figure 1 Constant Temperature Bath • • • • • • • • • • • 2 Effects of 2-chloroethanol Treatment on the Total Number of Ger.minated Allium Bulbs. • • 0 • • • • • • • • .. • • • • • 3 Effects of 2-chloroethanol Treatment on the Total Number of Germinated 4 5 Allium Roots .............. . Comparison of Mitotic Division in Roots of Allium cepa Obtained by Different Investigators • • • • • • • • • • • • • • • • • · . . . Effects of Temperature on Nuclear Division in Allium Roots. • • • • • • · . • • • 6 7 • Glass Solution Holder • • • • • • • • • Glass Solution Holders Suspended on the Wire Rack in the Constant Temperature Bath. • • • • • • • • • • • • • • • • • · . . . • • • • 8 Effects of Colchicine on Nuclear Division Page 11 23 24 45 50 56 56 in Allium Roots • • • • • • • • • • • • • • • • 65 9 10 Effects of Veriloid on Nuclear Division in Allium Roots .. .. • .. .. • • • • • •• Effects o~ Protoveratrine A on Nuclear Divis ion in "'Allium Roots 0 • • • • • • • • • • • 77 • • • • 85 I • GENERAL INTRODUCTION Many alkaloidal plant constituents demonstrate marked physiological activity when tested in warm-blooded animals. Extracts from the seeds o£ the plant Colchicum, for example, were said to be effective in the treatment of many human ailments and were employed in ancient materia medica long before the birth of Christ. References to the use of various seeds by Egyptian physicians for the treatment of aches and pains were made in the Ebers Papyrus (Bryan, 1931), probably our oldest medical text, prepared about 1550 B.C. Colchicum could ~ery well be one of the saffron plants mentioned in the Papyruso Plants referred to by Pliny as the hermodactyls (Chopra, 1933) were well known to the Hindu civilization (Majumdar, 1951) and have been identified as Colchicum luteum and Merendera persioa, both of which contain the alkaloid colchicine. In the first century A.D., Dioscorides wrote of an aut­umn- flowering crocus-like plant which grew in the land of Colchls, located on the eastern side of the Black Sea, and whence the plant derives its name Colchicum (Gunther, 1934). So careful were Dioscorides l scientific observations, des­criptions, and drawings that writers copied his work for fifteen centuries afterwardo By the nineteenth century, Colchicum preparations had established themselves in modern -2- therapeutics as specifics for the treatment of gout (Sharp, 1909; and Williwms, 1947). In 1820, Pelletier and Caventou isolated the toxic principle from Colchicum autumnale but, interestingly, they believed it to be veratrine, an agent later shown to evidence polyploidy. It remained for Geiger (1833) , to extract a distinct crystalline alkaloid which he sub-sequently nwmed colchicine. At the turn of the 20th century, Dixon and Malden (1908) reported that colchicine appeared to "excite karyo­kinesis" and therefore, these workers are usually credited with the first observation of the action of colchicine on mitosis. A prior paper (Pernice, l889), however, contained pictures showing arrested metaphases, although at that time, their significance was not entirely realized. The remarkable biological properties of colchicine were not fully revealed until the work of Lits was published in 1934. As a student working under Ao P. Dustin, Sr., Lite noted that colchicine caused a marked increase in the num-ber o~ mitoses when in contact with rapidly dividing, mer i­stematic tissues. Subsequent to the appearance of this work, a "veritable explosion" of publications occurred and firmly established colchicine as a true mitotic poison which interferes with the process of nuclear division and select- -3- ively inhibits the formation of spindle fibers thereby modifying the nuclear processes at metaphase (Brues, 1936; Brues and Cohen, 1936; Blakeslee and Avery, 1937; Eigsti, 1938; Kostoff, 1938; Levan, 1938; Bureau, 1939; Wellensiek, 1939; Dermen, 1940; King and Beams, 1940; Krythe and Well­ensiek, 1942; Vilter, 1944; Bucher, 1945; Ludford, 1945; Steinegger, 1946; Lehmann, 1947; Meyer, 1948; Levine, 1951; Cook and Loudon, 1952; Inoud, 1952; Eigsti and Dustin, 1955; and others). Thus, this chemical tool provided a new experimental approach to the study of plant and ani-mal cell division processes. As a result, a greater under­standing of the mechanisms involved in nuclear mitotic division and cytology has been achieved. Further, colchi­cine and other agents are now employed in medicine to study the complex mechanisms involved when normal tissues suddenl~' assume abnormal ~unctions and produce bizarre charlges, ~, the stUdy of numerous neoplastic disorders confronting medical science todayo Colchicine has also become an extremely use~l tool in botany (Avery and John­son, 1947). By treating seeds, root tips, and most plant parts where rapid meristematic activity resides, it has been possible to develop true chromosomal plant mutants much more rapidly than nature herself could produce them. According to Randolph (1941), the first production of experi- -4- mental polyploidy was achieved in mosses in 1908 by the Marchals using regeneration technics, and the first poly-ploid production in higher plants employing graft hybrid technics was induced in 1916 by Winkler. Polyploids may also be developed by chemical treatment with colchicine (Blakeslee and Avery, 1937; Blakeslee, 1939; Randolph, 1941; and Mangelsdorf, 1952); thus is provided an abundance of research possibilities for both the plant botanist and the commercial plant breeder. Many chemical agents other than colohicine have been used to produce nuclear and cytoplasmic ohanges in plants; chloral hydrate (Dreyfus and Zaccaro, 1933), caffeine and theophylline (Mangenot and Carpentier, 1944), propane and argon (Ferguson et al., 1950), coumarin (Cornman, 1947), chloroform (Steinegger and Levan, 1947), nitrogen mustards (Novick and Sparrow, 1949), acenaphthene (Fatalizade, 1939), sul.fonSmides (Traub, 1941), antibiotics (Wilson, 1950), benzene (Berger ~ al., 1944), and others. Also, physical factors have been used to produce nuclear and cytoplasmic changes in plants 0 For example, sudden changes in environ­mental conditions (especially temperature) may so alter cell division processes as to produce plant polyploids (Belling, 1925; Randolph, 1932; Dorsey, 1936; Sax, 1937; Dermen, 1938; Beasley, 1940; and Shimamura, 1940). However, environmental changes which produce plant polyploids are rather drastic, but they nevertheless have proved successful to a limited extent o In 1944, Witkus and Berger reported on "Veratrine, a New Polyploidy Inducing Agent". According to these authors the cytological effects of this agent 'were similar to those of colchicine with a few differences in the mechanism of action by which po1yp1Qidy was produced o The fact that active principles of Veratrum and related genera (~­denus) are effective in producing polyploidy is of interest, since the hypotensive effects of these plant extracts have been extensively investigated by the medical profession within the past few years. Because of this renewed inter-est, numerous investigators have attempted to isolate new Veratrum extracts (Saito et al o , 1934; Poethke, 1937; Poethke and Trabert, 1947; Poetsch and Parks, 1949; Hennig et al., 1951; Nanobashvili, 1951; and Kupchan and De1iwala, 1952), to characterize the isolated derivatives (Wright and Luff, 1879; Salzberger, 1890; Poethke, 1937; Craig and Jacobs, 1942; Jacobs and Craig, 1943, 1944; Fried et al o , 1949; Klohs et a1o, 1952; and Nash and Brooker, 1953), and to evaluate what, if any, pharmacological activity resides in the agents thus obtained (Richter and Thoma, 1939; Krayer and Mendez, 1942; Spitzy, 1949; Fried et ~, 1950; Meilman and Krayer, 1950; Klohs et al., 1952; Mosey and Kaplan, 1952; and Swiss, 1952). The number of alkaloidal constituents ob­tained from Veratrum vir ide Ait. alone has been estimated at approximately twenty. With this renewed interest in Veratrum viride and related derivatives as hypotensive agents, many purified alkaloids heretofore unobta~- able have now become available. Veratrine in reality is a mixture of alkaloids (Merck, 1855; Schmidt and Koppen, 1876; Blount, 1935; Youngken, 1948; and Wallis, 1955) obtained from sabadilla seeds (Schoenocaulon officinale A. Gray, Sabadilla officinali.s. Benth., Veratrum sabadilla Retz.) and consi~ts primarily 'of cevadine (crystalline veratrine) and veratridine (amorphous veratrine). Because of the close structural similarity existing between the alkaloids in veratrine and the alkaloids found in Veratrum viride Ait. and Veratrum album L., two reasonable questions may be asked: 1. Would alkaloidal mixtures or some purified alka­loids obtained from Veratrum viride and/or ~­trum album possess the ability to modify the mitotic cycle of nuclear and cellular division as does veratrine? 2. If so, how would such activity compare in kind and degree to that already so well established -7~ for colchicine? Before either of these questions can be answered, how­ever, one must know what effect other physical and chemical factors have on the nuclear division processes. It is well known that external environment exerts a marked effect on the growth of plants~ Since nuclear and cellular divi­sion are closely related to plant growth, it is reasonable to expect changes in the external environment to influence greatly these processes. Of the many factors which affect growth in plants, two of the most variable factors probably are light and temperature~ In addition, other factors, such as dor.mancy and root length, may influence nuclear division processes and present temporary obstacles to growth studies. Therefore, an investigation of these factors should provide much additional and perhaps very valuable information. In order to present adequately the effects of environ­mental changes, chemical agents, etc. on nuclear division processes, this dissertation has been prepared in three main parts: 1. The effects of preliminary tests on nuclear mitotic division in root tips of Allium cepa L. which are unrelated to environmental changes; 2. A study of the effects of environmental changes on -8· nuclear division and elongation, particularly of the effects produced by variations in light and temperature on this same plant tissue; 3. A study of the effects of two Veratrum alkaloids, Veriloidl and protoveratrine A2, on nuclear divi­sion and a comparison of these drug effects with the effects produced by colchicine. Veriloid and protoveratrine A are the chief alkaloidal con­stituents of Veratrum viride Ait. and Veratrum album L., respectively. standardized Veriloid has been available for same ttme but crystalline protoveratrine A was first reported in 1952 by Klohs, et al., and referred to as protoveratrine (as opposed to a closely related alkaloid, neoprotoveratrine). Nash and Brooker reported the isola­tion of the same alkaloid in 1953 and named it protoveratrine A, by which name it is now generally recognized. It was anticipated that such observations would provide further knowledge of the action of these Veratrum alkaloids and col-lSupplied by Dr. J. E. Campion, Riker Laboratories Inc., Los Angeles, Calif. Veriloid is a mixture of active prin­ciples from Veratrum viride Ait. standardized for hypo­tensive activity to reference standard alkavervir. 2Supplied by Dr. J. E. Campion, Riker Laboratories Inc., Los Angeles, Calif. and Dr. L. C. Weaver, Pitman-Moore Company, Indianapolis, Ind. protoveratrine A is a puri­fied alkaloidal fraction obtained fram Veratrum album L. Appreciation is expressed for the agents provided by the individuals representing these firms. -9- chicine on nuclear division and rurther elucidate the errects produced by changes in the external environment on nuclear division and root elongation. -10- II. GENERAL PROCEDURES Unless otherwise indicated, the following procedures were employed in all experiments. Onion bulbs, Allium cepa L. var. White Portugal, were used as the experimental plant. Beakers of 100 ml. capacity were filled with a solution of 2 per cent 2-chloroethanoll in tap water. Bulbs were placed in these beakers for 3 hrs. and the solu-tion level was adjusted so that the portions of the bulbs containing the root primordia were completely immersed in the liquid. The purpose of soaking the bulbs in the 2-chloro-ethanol solution was to induce the sprouting of more roots than would result from using tap water alone (see page 22). At the end of this 3 hr. soaking period, the bulbs were transferred to beakers containing only fresh tap water. The Allium bulbs remained in the tap water (the water was replaced each day) until roots sprouted, which was approxi­mately 1 to 10 days. During the tUne interval required for the Allium roots to sprout, the temperature of the water and the air was maintained between 25 and 300 C. After roots had sprouted, the Allium bulbs were placed in a constant temperature water-bath (Figure 1). The bath consisted of a circular "pryex" brand glass jar 12 in. high by 12 in. in lSupplied by Distillation Products Industries, Rochester 3, N. Y., division of Eastman Kodak Companyo -11- -12- diameter which was surrounded by a heavy semi-circular iron support. A rod shel~ suspended on adjustable chain hanger~ was located inside the water bath. The water was heated by immersed heaters consisting of insulated nichrome elements in flexible copper tubing. In order to obtain below room temperatures, the water was cooled by separate copper tubing coils through which cold water was circu­lated. By means of these heating and cooling coils, tem­peratures ranging from 10 to 1000 0. could be achieved. A "Merc-to-Merclt thermoregulator of the double meroury column type and a ItMerc-to-Merclt control box provided accuracy of temperature control to!D.5°0. A motor stirrer provided constant circulation of water in the bath for the entire duration of any given experiment. Bulbs'were placed on the rod shelf and water brought to the desired level on the bulbs. Roots from 1.5 to 6.0 cm. in length were used for all observations of nuclear division. Root tips, approximately 2.5 cm. long, were cut from germinating bulbs maintained at 300 0. during all experimental procedures, and prepared for microscopic examination by a modification of War.mke'B root tip smear method (1935). This method was devised for the rapid study of chromosome numbers in genera (Trillium, Allium, ~.) whose chromosomes are so large that the usual sectioning technics are inadequate. -13- Root tips were killed and fixed for 12 brs. or longer in a solution of 1 part glacial acetic acid and 3 parts absolute alcohol. Root tips have been shown to retain their cellular organization for two to three. weeks in this solution (Warmke, 1935). After they were killed, the roots were placed in a solution consisting of equal parts 95 per cent alcohol and concentrated hydrochloric aCid. A 5 to 10 min. soaking period was recommended by Warmke, however, it was found that soaking root tips for only 1 min. dis­solved the pectic substances of the middle lamella and did not affect the ability of the chromosomes to take stain. Despite the apparent severity of the strong acid solution, the 1:1 mixture has been shown not to distort chromosomes (Warmke, 1935). After this treatment, the roots were trans~ ferred to Carnoy1s solution (with chloroform)l for 3 min. to reharden the tissue after the hydrochloric acid treat-ment. The roots were removed from the Carnoyts solution and a small piece, approximately 2 mm. in length, was cut off the tip of the roots and placed in a depression on a spot plate containing 5 to 10 drops of Belling1s Iron-ace­tocarmine stain (Belling, 1926) and stained for 15 min. The stain was prepared according to recommendations given lCarnoyts solution chloroform • • • • • • • • • • • 3 parts absolute alcohol • • • • • • • • 2 parts glacial acetic acid. • • • • • • 1 part -14- by Johansen (1940)1. The small piece of root in 1 drop of the stain was then transferred to a clean glass slide, pressed firmly with a rubber spatula to separate the cells from one another, and a cover slip placed over the stain in which the cells were suspended. Gentle heating of the slide for a very short period without boiling the stain in order to clear the cell cytoplasm and thus provide bet-ter contrast to observe the nuclear material has been fre-quently recommended; however, this procedure was found to be unnecessary for this study since the original staining recommendations gave excellent results without heating. At this point in the procedure, if desired, slides could be permanently mounted and preserved by any acceptable method (McClintock, 1929; and Johansen, 1940), but because of the large numbers of slides needed for this study and the additional time required for such a procedure, it was deemed impractical. Cells were observed through a laborato~ micro- IBellingts Iron-acetocarmine stain (preparation recommended by Johansen). glacial acetic acid •• 0 • 0 • • • • • 90 ml. distilled water • • • • • • • • • • • • 110 ml. certified carmine dye • • • • • 0 • •• 1 gm. ferric chloride t.s., a sufficient quantity Add the glacial acetic acid to the distilled water, heat to boiling, and immediately add the dye. Cool in a refrig­erator, then decant. Add a few drops of the ferric chlor­ide test solution until the color on standing is a dark wine red. Do not add too much iron or the carmine will be precipitated. -15- soope at a magnification of 440 diameters (high dry), using a 100-watt artificial light souroe as the means of illumina­tion. The cover slip on each slide was divided by eye into thirds along the horizontal axis, an~ by means of a mech­anical mioroscope stage, one complete scan from one side of the cover slip to the other using only the horizontal oontrol knob was made through the upper, middle, and lower third. All intact oells observed under the cover slip in the three scannings, either resting or in any stage of mi-totio nuolear division, were recorded and the per cent of oells in nuolear division was oalculated. In order to analyze the experimental data obtained in this dissertation and draw oonclusions therefrom on a sta- I tistioal basis, suitable technics were employed (analysis of variance, ohi-square, and linear regression). The analysis of varianoe teohnio was developed b~ R. A. Fisher (1924) to faoilitate the handling of problems for test­ing hypotheses conoerning more than two normally distribu­ted populations. This technic "perhaps the most powerful and widely used statistioal technique" (Ostle, 1954), applied to a oompletely randomized deSign, was employed to interpret the data obtained in the individual experi-ments. In a number of instances, the data were assooiated with the binomial distribution: a certain number of ooour-rencee .. among a def:tli1te'total counted, expressed as percentage -16- of occurrences o In order to satisfy the assumptions which underlie an analysis of variance (Stearman, 1955), it was necessary in most exper~ents to perform a minor transfor­mation of the original data to angles before attempting the formal analysiso Tables for such transformations have been prepared and are available (Snedecor, 1946). By em­ploying the values transformed from the original data, the analysis of variance was then performed in the usual man­ner. The chi-square and linear regression technics were performed on only two exper~ents; therefore, the specific application of these technics will be discussed in detail in the sections where the exper~ental methods and results are described. -17- III. THE EFFECTS OF PRELIMINARY TESTS ON NUCLEAR DIVISION AND ROOT GROWTH IN ALLIUM A. The Effects of Chemical Treatment with 2-chloroethanol on Nuclear Division and Stimulation of Root Growth 1. Introduction I The growth stimulating influence of 2-chloroethanol (ethylene chlorohydrin) in plants is well known. In 1926, Denny reported that 2-chloroethanol facilitates the appear­ance of sprouts on dormant potato tubers. He subsequently demonstrated (Denny, 1937, 1938), that the resting period of Gladiolus corms could be effectively broken when they were treated with solutions containing 2-chloroethanol and that the treated corms would sprout and bloom before the untreated corms developed sprouts. Further, he found that 2-chloroethanol treatment would cause a 2-to 9-fold increase in the yield of young corms (1942). These obser-vations on potato tubers and Gladiolus corms have been confirmed by Michener (1941), Townsend (1941), Reichert (1941l.), and Denny (1938, 1942, 1943, 1945). The interest aroused by these studies resulted in the use of 2-chloro­ethanol in other specialized botanical fields. Thus, dormancy has been broken and germination effected in Jerusalem artichokes (Steinbauer, 1939), various genera of forest trees (Johnson, 1946), crabgrass (Gianfagna and Pridhwm, 1951, 1952), aged grain seeds, particularly oats (Rugge, 1947, 1952), and sweet potatoes (Michael, 1952). Since preliminary studies on Allium showed only 60 per cent of the bulbs germinated after two weeks incubated in tap water, it seemed important to investigate the influ-ences of 2-chloroethanol on nuclear division, on the num-ber of bulbs which sprout roots, and on the total number of sprouted roots as well as the rate at which these new roots developed. 2. Methodsl This study was divided into two parts: first, the effects of 2-chloroethanol on nuclear division processes; and second, its effects on the various fUnctions of root growth. In the first experiment, 16 Allium bulbs were randomi-zed into 3 groups, using a suitable Table of Random Numbers (Kendall and Smith, 1939; and Arkin and Colton, 1950). The 3 groups were: 1. bulbs soaked in tap water (control); 2. bulbs soaked for 3 brs. in a 2 per cent solution of 2-chloroethanol in tap water; ;. bulbs soaked for 6 hrs. in a 2 per cent solution! lFor details concerning technics employed for sprouting roots, microscopic preparation, cell counting, etc., see the section entitled General Procedures, page l~ -19~ - ~. of 2-chloroethanol in tap water. Roots were killed at the an~ of each test time, cells were observed, and the total number of cells in all stages of nuclear mitotic division was calculated and the ratio of this number to the total dividing cells present was expres­sed in per cent value. A comparison of the effects pro­duced on nuclear mitotic division in the 3 groups was per­formed by an analysis of variance (see page 15). A population sample of 100 bulbs was used in the second experiment. of 50 bulbs each. Bulbs were randomized into 2 groups One group of 50 bulbs was soaked in a solution of 2 per cent 2-chloroethanol in tap water for a period of 3 hrs., while the other group, serving as the con­trol, was soaked for the same period of time in tap water alone. At the end of 3 hrs., bulbs fram both groups were placed into fresh tap water only. All bulbs were then al­lowed to remain in the tap water, which was replaced fresh each day, until the conclusion of the experiment. The num­ber of bulbs sprouted and total number of roots germinated for both the treated and non-treated groups were counted and recorded daily for a period of 17 days, at which time the number of observed germinating roots had approached a plateau. In order to test the effects of the 2-chloroethanol treatment on stimulating a greater number of bulbs to sprout -20- roots, a 2 x 2 chi-square test of the data was performed at the point where the greatest difference occurred between the treated and the control groups. Differences in the total number and rate of sprouting of new roots between the two groups were statistically analyzed by calculation of linear regression (Snedecor, 1946) along the straight-line por-tion of the growth curve developed from each group. Both growth curves assumed essentially linear relationships during the period of the fifth day to the fifteenth day of the experiment. 30 Results The effects of 2-chloroethanol on nuclear division are listed in Table 1 from which it may be seen that only slight differences in the mean number of dividing nuclei were induced by this agent 0 The significance of the data presented was determined by the analysis of variance as shown in Table 2 and an e.xperimental F ratio calculated: F = 11.105/3.652 • 3.04. From a table of theoretical F values (Snedecor, 1946), FG~ .o~ = 3.80; since the experi­mental F value did not exceed the F value obtained from the table at the 5 per cent probability level, the experi­ment gave no significantl evidence of differences in nuc-lear division between times of exposure to the 2-chloro- IThe terms "significantn and "highly Significant" will be used hereafter to denote the existence of statistical differences at the .05 and .01 significance levels, respectively. -21- Table 1 Estimates of Nuclear Division Means from 2-chloroethanol Effects T2;i(m e2 -ochf lEorxopeotshuaren otlo DMiveaidn in%g Standard Nuclei Error Control 3.3 11./. 41/6 3 Hrs o 4.1 V.4l/5 6 Hrs. 2.3 V.4l/5 Table 2 Analysis of Variance'" of 2=chloroethanol Effects on Nuclear Division in Allium Roots Adjustment for Hean 1696062 Nature of Variation DegFees Sum of Freedom Squares Between exposure times 2 22021 Error 13 47.4$ Total 15 69.69 +Transformation of data to angles. = = = .26 .28 .28 Mean Square 11.105 30 652 -22- ethanol treatment of the roots. The results of the second experiment, the effects of the 2-chloroethanol on the number of germinated bulbs and roots, are shown in Figures 2 and 3. An examination of the growth curves in Figure 2, revealed little difference between the 2-chloroethanol treated group and the control group. The greatest spread between the two curves occur­red on the fifth and sixth days after the start of the experiment. A simple 2 x 2 chi-square test between the number of germinated bulbs and the number of non-germinated bulbs observed on the sixth day of the exper~ent for both the control and the 2-chloroethanol treated groups revealed that even a difference in sprouting of 5 Allium bulbs was far from significant; experimental chi-square: 0.71; chi­square for 1 degree of freedom = 3.84. Treatment with 2 per cent 2-chloroethanol, therefore, failed to stimulate germination of a significantly greater number of bulbs than was produced by tap water alone. A comparison of the results obtained from the 2- chloroethanol treatment on the total number of germinated roots is shown in Figure 3. Points representing the total number of germinated roots for each test group from the fifth to the fifteenth day of experiment were plotted. Linear regression equations were calculated by the method of least squares, and straight lines were then drawn thro~gh 50 40 5 ~ 30 &3 f-< ~ ~ t:? o:! [;il CJ 20 ~ ffi I 10 o I,..!!I' 1 2 3 "",' " , " "D" Ji',,'-' 4 5 6 7 8 9 DAYS 0--------0 z1; 2-chloroethanol treated o () Tap water (control) _... . 10 11 12 13 14 ,. - ..,q''''' 15 ..-..­p---- o 16 17 Fig. 2. Effects of 2-chloroethanol Treatment on the Total Number of Germinated Allium Bulbs. i N Lu I 1100 1000 900 800 en E... . 0 700 0 p::; ~ E-< 600 <t; :z; ~ 500 t'J f;l:., 0 &1 400 §! ~ JOO 200 100 0 9/ 1 2 J 4 5 6 7 8 9 DAYS c- - ------0 'i!!j; 2-chloroethanol treated o 0 Tap water (control) / / ,/ 10 / 9'/ / 11 / /c / / 12 / / / /c lJ '" '" '" '" /~ 14 '" C / / 15 16 Fig. J. Effects of 2-chloroethanol Treatment on the Total Number of Germinated Allium Roots. 17 I I\) .f="" B -25- points calculated fram the equations. In the 2-chloro­ethanol treated group, new roots sprouted at a constant rate of approx~ately 82 roots per day from the fifth to the fifteenth day of the experiment, (slope value, b = 82.34), whereas in the non-treated (tap water) group, the rate of new root development was approximately 67 roots per day (b = 66 0 66)0 Calculation of the standard devia­tion from regression (standard error) and the fiducial limits at the .05 level of probability (95 per cent con­fidence limits) gave values of 82.34 (78 .. 72 - 85.96) roots and 66.66 (62.26 - 71.16) roots for the 2-chloro­ethanol and tap water groups, respectively. Further, a ftStudent ll t test comparison indicated a highly signifi­cant difference existing between the two slopes; experi­mental nStudent ll t value = 9081, t( .. Ol) for 9 degrees of freedom • 3.250 The existence of such significant differ­ences may also be noted by the non-overlapping of the fidu­cial limits.. These results indicate, therefore, that in bulbs of ~llium cepa treated with 2 per cent 2-chloroethanol, the rate of appearance of new roots was significantly in­creased over the rate observed for bulbs soaked in tap-water alone." 40 Discussion Although the effects produced by the 2 per cent solu- -26- tion of 2-chloroethanol for 3 and 6 hrso exposure were not significantly different fro~ results obtained for the control (see Table 1), there appeared to be some observable effect produced by the agent on the nuclear division processes o For example, further examination of Table 1 suggests that the 3 hr. exposure to the 2- chloroethanol solution brought about about a slight increase in the number of dividing nuclei beyond that noted for the control groupo Further, the six-hour exposure time for the 2-chloroethanol appears ·to have had some inhibitory effect on nuclear division. Not only was the number of dividing nuclei markedly less for the 6 hr. exposure time (2.3 per cent) than for the 3 hr. period (4 0 1 per cent), but it was also less than the value calculated for the control (3.3 per cent)o It is interesting to note that the 3 hr. exposure to the 2-chloroethanol appears to have stimulated nuclear division, albeit only slightly. Be­cause of the increased rate of root germination due to the same treatment (Figure 3), possibly the number of divid-ing cells in each root tip also increased above normal and contributed to all or a portion of the observed increase in Allium root germination rate. Three possible explanations for the failure of the 2~chloroethanol treatment to increase the number of ger­minated bulbs from that observed In the control are immedi- ately apparent: 1. 2-chloroethanol is incapable of breaking dormancy in the variety of Allium (White Portugal) employed in the experimento From the results observed for the effects on root growth (Figure 3) and the re­sults reported in the literature for other plant genera, this explanation is probably the least plausible 0 2. The bulbs employed in the experiment were incap­able of germinating under any circumstances; there­fore, the 2-chloroethanol could not be expected to have been any more effective than it was. 3. The concentration and time of exposure employed for the 2-chloroethanol was inadequate to break dormancy in the non-germinated bulbs. The finding that a ·2 per cent solution of 2-chlbroeth­anol effectively stimulated the rate of germination of new roots (Figure 3), whereas it failed to have any effect on the number of germinated bulbs in the same experiment, in-dicated that the drug was effective to a limited degree. These findings are in agreement witb the literature reports (De~?37, 1938; Townsend, 1941; Michael, 1952; and -"-' .. ~~--:-- others) on the effect of 2-chloroethanol in plants. Since the 2-chloroethanol in 2 per cent concentration for a 3 hr. exposure time increased the daily germination rate of new roots by approximately 19 per cent over control values, there remains the possibility that this strength may not have produced the optimum effect; a differ>ent drug concen­tration or time of exposure might have produced even more striking results o These and related problems with 2-chloro­ethanol in Allium bulbs are as yet unanswered, but the experimental results reported herein suggest possible new avenues for further fruitful studyo -29- B. The Relation of Root Length to Nuclear Division 1. Introduction In the course of investigating the various environ­mental and drug induced alterations on nuclear division in Allium bulbs, there appeared to be a relation between root length and the number of cells undergoing mitotic nuclear division. For example, newly sprouted roots, less than about 3 cm. in length, appeared to elongate approxi­mately twice as fast in the same period of time as roots which were more than 3 cm. long. Inasmuch as the rate of elongation in the Allium roots decreased as the roots became longer, it was considered important to determine whether the total number of cells undergoing mitotic nuclear division would vary as the Allium roots increased in length. 2. Methods A population sample of 75 bulbs was used in this study. The sample was randomized into 15 groups of 5 bulbs each, and the bulbs were placed in water and roots were allowed to sprout in the usual manner (see page 10 ). When the roots from the 5 bulbs in group one measured 0.1 to 0.5 cm. in length, they were cut, killed, and prepared for microscopic examination of the cells; when the roots in -30- each of the other groups reached the desired length (roots in group 15 measured 7.1 to 7.5 cm. in length) they were also cut, killed, and fixed far microscopic examination. A comparison of the effects produced on nuclear mitotic division in each of the 15 groups was performed by an analysis of variance (see page 15). 30 Results The effects of different root lengths on nuclear division are listed in Table 30 The numbers of dividing nuclei appear to increase as roots became longer. The experimental ratio calculated from Table 4 was F = 80 578/ 40728 = 1.81; from th~ table of theoretical F values, F li(~ .o~ = 1 0 86. Since the experimental F value did not exceed the F value obtained from the table at the 5 per cent probability leve~, it suggests that there is no significant difference in the number of dividing nuclei in roots up to 705 cm. in length. Because the figures obtained for the experimental and 5 per cent level F values were numerically nearly equal, the experiment was repeated and the results are listed in Table 5. However, the root length groups are in 1 cm. increments rather that in one-half cmo increments as in the previous experiment (Table 3)0 As in the previous experiment, the number of dividing nuclei appear to increase as roots become longero The experimental ratio for this experiment ~31- Table 3 Estimates of Nuclear Division Heans for Various Hoot Lengths Root Length Mean % Standard Dividing I (em. ) Nuclei Error 0.1 to 0.5 2.4 " 0.6 11 1.0 2.6 101 II 1.5 3 .. 2 106 " 2.0 2.6 2.1 " 2.5 3.1 2.6 II 3.0 3.5 3.1 II 3.5 4.0 Y.68/5 3.6 " 4.0 4.2 4.1 " 4.5 4.1 4.6 II 5.0 3.7 5.1 II 5.5 3.4 5.6 11 6.0 5.3 6.1 11 6 .. 5 3.7 606 " 7.0 4.9 7.1 H 7.5 408 Table 4 Analysis of Variance+ of Root Length Effects on Nuclear Division in Allitur. Roots Adjustment for Mean 8987.43 Degrees Sum of Nature of Variation Freoefd om Squares Between different root lengths 14 120.09 Lrror 60 283.66 Total 74 403.75 +Transformation of data to angles. = .. 37 Mean Square 8.578 4.728 -32- Table 5 Estimates of Nuclear Division Means for Various Hoot Lengths Root Length DMiveaind in%g Standard (em .. ) Nuclei Error 0 .. 1 to 100 2.1 ~ 1.1 II 200 463 2 .. 1 II 300 4 .. 3 ~1.7/6 301 " 400 • 4.8 4.1 II 500 4 .. 7 5.1 II 6.0 • 3.7 I .... Table 6 Analysis of Variance+ of Root Length Effects on Nuclear Division in Allitun Roots Adjustment for Mean 4707 .. 56 DeaFees Nature of Variation Sum of Freedom Squares Between different root lengths' 5 77.47 Error 30 224.65 Total 35 302 .. 12 +Transformation of data to angles .. = .53 Mean Square 15 .. 494 7.488 -33- calculated frem Table 6 was F = 150494/7.488 = 2.Q7; the theeretical F value was F [;g .o~ = 2053~ Again, as in the previeus experiment, there was no. significant diff-erence between reet length and nuclear divisien. It ceuld be assumed, therefere, that reets ef any length up to. 7.5 cm. leng may be empleyed by this experimental precedure fer studies en the number ef dividing nuclei in Allium. 40 Discussien The data presented suggest that, altheugh the num-ber ef dividing cells in roets ef different lengths are net significantly different, reet length has seme effect en the nuclear divisien precesses. Fer example, examin-atien ef Tables 3 and 5 indicated that the number ef divid­ing nuclei was censistently lew (appreximately 2.4 per cent) in very shert reets from 0.1 to. 1.0 cm. in length fer beth the eriginal and repeat experiments. The eppesite might be expected to. eccur if the rate ef mitetic nuclear divi­sien is assumed to. influence the rate ef reet grewth (in length) directly. Reets with rapid grewth rates, by the assumptien just made, weuld have a high number ef mitetic dividing nuclei fer any given peried ef time. It weuld appear then, frem the results ebtained in these experi­ments that Alli~ reets (up to. appreximately 1.0 em.) initially increase in length mere frem cell elengatien than frem the preductien ef new cells in the meristematic -34'· root-tip region by mitotic nuclear division. Kellicott (1904) and Friesner (1920) reported the existence of a reciprocal relationship between elongation and cell divi­sion in roots of Allium cepa o The number of dividing nuclei for roots longer than 1.0 cm. assumed a relatively constant rate of nuclear division (approximately 4.0 per cent) at any given time; therefore, in order to minimize the influence of root length on nuclear division as an experimental variable, short roots less than 1.3 cm. in length were not used. For further observations of mitotic dividing nuclei, only roots from 1.5 to 6.0 cm. in length were employed. IV. THE EFFECTS OF ENVIRONMENT ON NUCLEAR DIVISION AND ROOT ELONGATION IN ALLIUM A. The Effect of Light on Nuclear Division 10 Introduction Variations in exposure to light constitute some of the most important factors which influence growth in plants. Daily periodicity in the elongation of aerial plant parts grown under natural environmental conditions has been known for many years o For example, Sachs (1872) published the results of his study on the periodic elongation of the stem in various plants; and the periodicity of leaf growth with different times of illumination was later reported by Prantl (1873), Stebler (1878), and otherso The effects of light and dark on nuclear and cell division processes in aeria.l and subterranean plant parts have been reported by Lewis (1901) and Karsten (1915, 1918)0 According to Friesner (1920), Kellicott, in 1904, experimenting wi th the roots of Allium was the first to note any regula.rity in elonga- ;) tion of roots grown under constant environmental (light and temperature) conditions 0 Thus, it has been established that natural alterna­tions of light and darkness induce a periodicity in elonga-tion, nuclear division, and cellular division; an effeot which gradually disappears when these conditions are ren- -36- dered constant. From the work of Kellicott (1904), Karsten (1915) and Friesner (1920), it was further demonstrated that under uniform conditions, there occurred a cell divi­sion rhythm.. This term was used to denote regular fluc­tll. atttms'1n-cell division rat.e under constant environmental conditions during each 24 hr. ,period, as distinguished from the term periodicity, used to describe more pronoun­ced fluctuations in the rate of cell division induced by environmental changes. Parallel findings from investiga­tions employing animal tissues have recently been pub­lished (Cooper and Schiff, 1938; Cooper and Franklin, 1940; and Blumenfeld, 1944). Because roots are frequently exposed to variations in exposure to light when grown by hydroponics, it was thought important to determine the effects of alterations in light and dark exposure periods on nuclear division and root length in this species of Allium. 29 Methods The study was divided into 3 experiments: 1. A study of the effects of time of day on nuclear mitotic division; 2. A study of the effects of changes in time of expos­ure to light on nuclear mitotic division; and -37- 3. A study of the effects of changes in time of exposure to light on root length. In the first experiment, a population sample of 40 Allium bulbs was randomly divided into 8 groups of 5 bulbs each. The groups were divided so that roots could be ob­tained at 3 hr. intervals over a 24 hr. period. Roots were killed at the end of each test time; cells were ob­served; the total number of cells in all stages of nuclear mitotic division was calculated, and the ratio of this number to the total cells present was expressed as a per cent value. A comparison of the effects produced on nuc­lear mitotic division in the 8 groups was performed by the analysis of variance technic (page 15). Since variations in the time of day involve changes in exposure to light, tte second experiment was designed to test the effects on mitotic nuclear division processes of changes in the time of exposure to artificial tungsten light adjusted to provide intensities on the roots equiva­lent to normal daylighto For purposes of standardization, normal daylight was defined as that intensity of light present in the laboratory during the daytime, which, by means of a standard photoelectric exposure meter, produced "'-'-iiilnimum and maximum readings of 13 and 40 foot-candles, respectively. A population sample of 16 Allium bulbs was randomly divided into 3 groups; each group was exposed to the light for different periods of time: one group was exposed for 72 hrs. to continuous light; the second group, the control, was exposed for 72 brs. to intermittent light (12 brs. light and 12 brs. darkness); the third group was exposed for 72 brs .. to cortinuous darkness. At the end of the 72 hr. test period, roots were killed; cells were ob­served; and the ratio (expressed as a per cent value) of the mitotically dividing nuclei to the total number of cells was calculated. A comparison of the effects produced on nuclear mitotic division in the 3 groups was again performed by the analysis of variance technic. The third experiment was designed to test the effects of changes in the time of exposure to light on root length. Sixteen Allium bulbs were randomly divided into 3 groups, each group having different times of exposure to the arti­ficial, constant intenSity light source. The different light exposures for the 3 groups were the same as that described in the preceding paragraph for 72 hrs.; continu­ous light, intermittent light, and continuous darkness. Roots were measured (in cm.) at the beginning of the experi­ment and at the end of the 72 hrQ test period. Changes in root length over the test time studied were recorded and a comparison of the effects produced on root length was per­formed by an analysis of variance. -39- 3" Results The effects of different times of day on nuclear division are listed in Table 70 The mean values suggested slight periodic fluctuations in the number of dividing nucleic However, statistical analysis of the data, as shown in Table 8, indicated that the number of mitotic dividing nuclei was not significantly changed for different times of day, the experimental F ratio = 20939/30517 = 0.84; theoretical F ~~ .~ = 2.32. The effects of continuous light, intermittent light, and darkness on nuclear division over a 72 hr. experimental test period are listed in Table 9. The mean values indicated that light may have an effect on the number of dividing nucleic Statistical analysis of these data revealed that changes in time of exposure to the artificial light employed in this experiment produced same changes, although again, not significant, in the mean number of dividing ~uclei since the experimental F value, F = 150730/4.361 = 3.61 approached the theoretical F value very closely at the 5 per cent probability level, F ~3 "o~ :: 3.80" The mean number of dividing nuclei (Table 9) was 107, 302, and 3.2 per cent for exposure to continuous light, intermittent light, and darkness (see paragraph 1, page 38), respectively. The effects of continuous light, intermittent light, and darkness on root length are listed in Table 11. Examination of the mean root lengths suggest marked differences due to -40 Table 7 Estimates of Nuclear Division Means for Different Times of Day Mean % Time of Day Dividing Nuclei 12:00 midnight 4.8 ..... 3:00 a':m. 5.2 6:00 a.m. 5.2 Standard Error 9:00 a.m. 4.5 >-V.38/5 = .28 12:00 noon 4.1 3:00 p.m. 5.4 6;00 p.m. 3.9 9:00 p.m. 5.2 '" Analysis of Variance+ of Time of Day Effects on Nuclear Division in Allium Roots Adjustment for Hean 6360.74 Degrees Nature of Variation of Sum of Freedom Squares Between different times 7 20.57 Error 32 112.54 Total 39 133.11 +Transformation of data to angles. Mean Square 2.939 3 .. 517 -41- Table 9 Estimates of Nuclear Division Means For Light Changes and Darkness Treatment Drii.lveaind in%g Standard Nuclei Error Continuous Ught 1.7 ~ 058/6 = Interr:l.H tent lirpt (control) 302 A.J 058/5 = Darkness 3.2 ~ 05~/5 = Table 10 Ana]ys:i s of Varia.nce l of the f,;i'fects of Changes in !',xposure to LiRht on Nuelear Ui vision in I.IJlum Hoots Adjustment for i-lenn 1359.77 TJe§Fees Nature of Variation Sum of Freedom 30 uares Bet",-een light concii tj ons 2 31 .46 Error 13 56.69 Total 15 88.15 +Transformation of data to angles~ .31 .34 .34 ;"lean Souare 15.730 4.361 Table 11 Estimates or Root Length Means for Light Changes and Darkness Mean Root Treatment Length Standard (em. ) Error Continuous light 1.3 V.95/48 Inte~ctottnetrno~l) light 1.6 Vo95/28 Darkness 3 .. 8 ~ .95/20 Table 12 Analysis of Variance of the Effects of Changes in Exposure to light on Root Length in Allhnn Roots Adjustment for Mean 344.3 Degrees Nature of VariaUon of S'lIDl of Freedom Squares Between light conditions 2 67.0 Error 93 88.5 Total 95 155.5 = = = .14 .18 .22 Mean Square 33050 0.95 -43- the light and dark exposures o Statistical analysis of these data, as shown in Table 12, revealed that light exposure induced highly significant changes in the mean root length, experimental F ratio = 33.50/0.95 = 35.26; theoretical F g oO~ = 4.82. For example, by noting the values for mean root length listed in Table 11, it may be seen that the mean length was 1.3 and 1.6 cm. for roots exposed to light equivalent in intensity to normal daylight for 72 hrs. (continuous light) and for roots exposed to intermittent light for 72 hrs. (12 hrso light and 12 hrs. darkness), respectively. This change was not significant when compared by a flStudent" t test for differences between two means (P).30), whereas, the mean length of roots exposed to darkness for 72 hrs. and for roots exposed to the intermittent light was 3.8 and 1.6 cm., respectivelyo These differences were Shown to be highly significant (P ( .. 001)0' 4 •. Discussion The data presented indicate that exposure to light has noticeable effects on mitotic nuclear division and frequently has marked effects on root lengtho Prolonged exposures to the artificial light employed in these experiments, as listed in Tables 9 and 11, apparently had an inhibitory effect on the number of mitotic div-iding nuclei and on the root length. Further, the marked inhibitory effect of light on root length, as compared with its observable but statistically non-significant effects on -44- mitotic nuclear division, is in agreement with the accepted belief that nuclear division and root elongation are separately functioning processes. Further evidence in support of this concept is the fact that recently sprouted, very short roots elongate rapidly, but have a low nuclear division rate (Tables 3 and 5)0 As previously pointed 'out, many investigators have reported daily fluctuations in the number of dividing cells for specified periods of time (see page 35) with maxima and minima occurring with remarkable regularity. Of interest is a graphic comparison of mitotic division in the roots -of Allium obtained by plotting the values from this study with results reported by, Friesner in 1920 using the same plant (Figure 4). The upper curve shows the data obtained from Table 7 for a 24 hr. period of time while the lower curve is derived from the work of Friesner (1920). It may be seen that at the sixth hour of the experiment, both curves reached their greatest maxDrrum and ~ediately afterwards fell to a low minimum. From the ninth to twenty-fourth hours, there was an overall, gradual increase in the divi­sion rate approaching the level seen at the start of the experiment 0 Although the fluctuations observed by Friesner (lower curve) were of much greater magnitude that those observed in this study (upper curve) it is of interest to note that" Friesner's results were apparently based on 6 H W ...:l u 5 '-' Z d z- 4 H....:l o ~ H CI> ~ () o s.. 3 IL.&: 0- t5 2 ~ ~ 1 --i2 300 ~ 250 ~ u d 200 z H 0 H pH . 150 0 IL. 0 &l 100 ~ z 50 0 3 Fig. 4. 6 9 12 15 18 21 24 TIl/JE (Hrs.) Comparison of ¥~totic Division in Roots of Alli~ cepa Obtained by Different Investigators. Upper Curve, Results Plotted from Table7l. Page ~O; Lower Curve. Results Reported by Friesner (1920). E ~ ! means calculated from roots of only 3 Allium bulbs; whereas, the upper curve of Figure 4 was drawn from the experimental means calculated from a population sample of 40 bulbs, and the results were shown to be non-s'ignificant at the .05 probability level (see page 39)0 As Friesner pointed out, the time of maxima and minima was dependent upon the time of initiation of metabolic activity of the roots. The times of root initiation were unknown for the experiment depicted in the upper curve in which a large population of bulbs was employed; therefore, the low number of fluctu­ations in mitotic division was expected. The use of an even greater number of bulbs (approximately 100) in future experiments of this nature might reasonably be expected to produce even fewer fluctuations over a 24 hr. period of time. The number of mitotic dividing nuclei for roots exposed to different times of exposure to light was not significantly altered o Because the ratios obtained between the experimental and theoretical F values (see page 39) were consistently close, it was thought important to main­tain exposure of roots to light of a const'ant intens i ty. For this reason, all subsequent experiments on Allium roots were performed using a constant light exposure (artificial tungsten light source) adjusted to produce a photoelectric exposure reading ranging from a min~ of 13 to a maximum of 40 foot-candles of light during the experi­mental periods. -47 B. The Effects of Temperature on Nuclear Division 1. Introduction Extremes in temperature have been shown to produce polyploidy in plants. In 1925, Belling demonstrated that heat shock was successful in producing triploid and tetra­ploid plants artificially; later, Randolph (1932) reported that by placing seedling root tips of maize in hot water (40 to 45°C.) for a period of 15 min. to 2 brs., he was able to achieve limited polyploid production. Stmilar uses of high temperatures have also been reported to induoe this effect in wheat, rye, and cotton (Dorsey, 1936; and Beasley, 1940). It appears that temperature extr~es, in general, are capable of modifying cellular aotivity, sinoe Sax (1937) found that the best results in modifying nuclear and oellular division in the plant Tradescantia oould be achieved by alternating the temperature fram oold (Boc.) to warm (38°C.) for a period of several weeks. Barber and Callan (194·3), employing cold termperature treatment (30 C.), observed arrested mitoses in dividing epidermal oells of animal tissue (newt). In view of the alterations in cell division prooesses known to occur with changes in temperature, it seemed ~port­ant to determine the effects of temperature changes on nuolear mitotio processes in the root tips of Allium in order to as- -48- certain what variations in temperature fram the 30oC. control could be permitted for future experiments and still not produce undesirable alterations in the nuclear mitotic activity. 2. Methods A population s~ple of 77 Allium bulbs was employed in these experiments. The bulbs were randamly divided into 7 main groups of approximately 11 bulbs each. The 7 main groups were further divided into 2 sub-groups of 5 or 6 bulbs each. Roots were exposed for 24 hrs. to 7 tempera­ture gradients fram 15 to 45°c. in increments of 5°c. The results obtained for each temperature tested were compared with those obtained in control groups with roots exposed to a constant temperature of 30oC. All roots were subsequently killed and fixed at the end of the test time; cells were observed; the total number of cells in all sta­ges of nuclear mitotic division was calculated; and the ratio of this number to the total cells present was expres­sed as a per cent value. A statistical comparison of the effects produced on mitotic nuclear division in each of the 7 main groups by the test temperatures as compared with the 30oC. control was performed by the analysis of variance technic. -49- 3. Results The effects of 7 different temperature gradients compared with their individual 300 0. control values after 24 hrs. are shown in Figure 5. The mean values for the test temperatures are represented by the plain vertical bars and the control mean values are shown by the slant­lined vertical bars; the fiducial limits at the 5 per cent probability level are represented by the vertical bracketed lines. Mean values of 3.5, 5.1, 5.2, and 4.0 per cent obtained from exposure of the roots to tempera­tures of 20, 25, 30, and 3500. were not significantly different from means of 5.7, 4.5, 3.5, and 4.5 per cent for the respective control temperatures. In contrast, means of 1.4, 1.6, and 0.20 per cent obtained for temperature extremes of 15, 40, and 45°0. differed sig­nificantly (40 and 450 0. were highly significant) from means of 3.1, 3.9, and 3.3 per cent obtained for the respective controls. 4. Discussion The significant decrease in the number of mitotic dividing nuclei at temperature extremes of 15, 40, and 450 0. emphasized the important role played by this environmental factor on nuclear mitotic division. The 8 7 6 5 g , ...... u ~"' ;~ 4~ ;Z:....:> H C Q (l) H U ~ ;... Q ().) ~l..c ;~ '-../ J 0 0:: >xl ::L: ;;,.; 'Z"'"' 2 1 I til l-LtN \11 I ~ Control {Jaoe.) D Test r+tN l, 01 11'\1 '''I 11),1 11'\1 1'\1 1'\1 1'1 15°C. 20°C. 25°C. looe. J50C. 40°C. 450C. TEST Fig. 5. E:'fects of Terr.p':!rature on Nuclear :Jivision in J.lli.UlTi Roots. B \Jl 0 I -51- mean values for the temperature extremes of 15, 40, and 45°c. (Figure 5), were approximately 55, 59, and 94 per cent less than their control means, respectively. It may be seen from this study that changes in tem­perature may produce marked alterations in nuclear mitotic activity in Allium roots; however, temperatures in the range permitting optimum physiological plant activity from 20 to 350 c. apparently do not produce undesirable alterations in mitotic function. Even though rather wide temperature fluctuations could be permitted in the media immediately surrounding the Allium roots without altering mitotic nuclear activity, in order to eliminate the effects of temperature as an experimental variable, it seemed import­ant to maintain the temperature as constant as possible (30oC.+0.5°C.) for the subsequent experiments in which drugs were to be tested for their effects on the mitotic processes. -52- V. THE EFFECTS OF LILIACEOUS ALKALOIDS ON NUCLEAR DIVISION IN ALLIUM ROOTS A. The Effects of Colchicine on Nulcear Division 1. Introduction In 1934, Lits described a drug method for the experi­mental alteration of the processes of cell division which was characterized by metaphasic arrest. This report, prepared under the direction of Dustin Sr., his senior professor, described the abilit~-of colchicine to cause a marked increase in the number of mitoses in rapidly dividing, meristematic tissues. The remarkably low effect­ive concentration (colchicine has been shown to be effect­ive in ooncentrations ranging from 1:100 to 1:10,000) and the number of arrested metaphases in a given treated tis­sue made the effects produced by colchioine very impres­sive. Later, it was established that colohicine aoted upon mitosis in both animal and plant tissues (Dustin!! ~, 1937). Living cells respond almost universally to colohicine after a basic pattern. This pattern consti­tutes the colchioine mitosis (abbreviated c-mitosis), in which seleotive inhibition of spindle fiber for.mation inter­feres with nuclear division at metaphase. A report describing the significance of multiple chromosome sets in Allium root tips was published by Gav- audan and associates in 1937, and called attention to poly­ploidy induced by colchicine. These investigators were aware of the cytogenetic implications introduced by the use of this drug. Wide interest in colchicine developed among botanists as the exper1r\lEUtal value of, this drug be­came clear. Beasley, in 1940 found that one out of 600 cotton plants treated by the temperature-shock technic (see page 47) became polyploid (1:600), but the colchicine procedures applied to a similar group yielded 50 polyploids fram among 100 (1:2) of the cotton plants surviving the chemical treatment. Eigsti and Dustin (1955) in their book, Colchicine--in Agriculture, Medicine, Biology, and Chemistry, made this statement: There are several noteworthy features of colchicine that account for its effectiveness as a polyploidizing agent. Briefly, colchicine is highly soluble in water; colchicine is not toxic to plant cells even in strong dosages; colchicine is effective in concentrations rang­ing from 1.0 to 0.01 per cent (1:100 to 1:10, 000); and finally, it is soluble in lipoids. Further.more, the effect obtained during a treat­ment is wholly reversible. Thus the drug is almost "made to order" for changing diploids into polyploids. Since colchicine has become the agent of choice for the exper~ental alteration of cell division processes, it seem­ed important to investigate the effects of this drug on nuclear activity in the roots of Allium cepa in order to establish a standard for subsequent studies on Veriloid and -54- and protoveratrine A. 2. Methods A population sample of 100 Allium bulbs was randomly divided into 5 main groups of 20 bulbs each. Each of the main groups was fUrther randomized and subdivided into 4 subgroups of 5 bulbs each. The roots on the bulbs in 4 of the main groups were treated with one of the following ooncentrations of colchicine: 0.1, 0.2, 0.4, or 0.8 per cent. The remaining main group served as a control. The roots of the 4 subgroups were exposed to the requisite colohicine solution for a predetermined period of time, .i......e......, 3, 6, 9, or 12 hrs. For example, the 20 bulbs in the main group treated with a 0.1 per cent solution of colchicine were subdivided into 4 subgroups of 5 bulbs each. The roots on subgroups 1, 2, 3, and 4 were exposed to the colchicine solution for 3, 6, 9, and 12 hrs., respectively. The plant Colchicum contains many alkaloids closely related to colchicine. One of these desmethyloolchioine, has been reported to be present in Colchicine U.S.P. (Horowitz and Ullyot, 1952); furthermore, colchicine solu­tions are susceptible to breakdown by the action of light and oxygen. Many reports on colchioine have been olouded by the lack of specification of the' source, method of prep­aration, or precautions observed with the use of the agent. -55- To clarify this confusion, Eigsti and Dustin (1955) made the following statement: The ~portant poin~ is that each paper should mention clearly the origin of the colchioine, whe­ther crystalline or not, whether purified and how, the method of preparing the solutions before the exper~ents, and the temperature at which these are oonducted. It is' only in this way that a valid oomparison of results is possible. For these reasons, the source and prooedures employed with the colchicine used in this studyare'listed: 1. Colchicine U.S .. p.l .in the form of a pale yellow, amorphous powder was the agent employed; 2. Fresh solutions, prepared by dissolving the requisite amount of drug in tap water to produoe the desired concentration, were used through­out the study; 3. As stated in previous ,sections (see pages 12 and 51 ), all exper~ents were conducted at a tem­perature of 30oC.+O.5°C. for the solutions im­mediately surrounding the Allium roots. Since the alkaloids were available only in very limited quantities (less than 1 gm. in some instances), special glass holders for the bulbs and alkaloidal solutions were oonstruoted whioh would hold approximately 2 to 4 mI. of solution (Figure 6). Eaoh glass holder consisted of a "Pyrex" brand watch glass (65 ImIl. in diameter) to which a piece of IIPryex" glass lManufactured by the Inland Alkaloid Company, Tipton, Ind. Fig. 60 Glass Solution Holder. : """""" "'- ...- ~ r:;:....- ~ ~l """"'" \~ _J ~~ L ....., Fig. 7. Glass Soluti.on Holders Suspended on the Wire Rack in the Constant Temperature Bath. -57- tubing (10 to 12 Mm. inside diameter) was heat-fused per­pendicular to the center on the convex side. By using vacuum suction on the glass tubing at the instant it was fused to the watch glass, a hole was made in the watoh glass. Then construction of the solution holder was com­pleted by sealing the opposite end of the glass tube app­roximately one and one-half inches from the fusion point. The holders were suspended on the wire raok (Figure 7) in the constant temperature bath and the ciroulating water around the holders maintained the alkaloidal solutions at the desired temperature (300 0.). A statistical comparison of the effects produced on nuclear mitotic division by different ooncentrations of colchicine and by different durations of exposure to the agent was performed by means of a speoialized form of the analysis of variance technic known as factorials. Factor­ial analYSis of the data to be presented per.mits the evalu­ation of the effects of time and of concentration independ­ently. In addition, information may also be obtained as to whether the 2 factors (time and ooncentration) may pro­duce effects which are interrelated. 3. Results The effects of different concentrations of colchicine and different t~es of exposure on nuclear mitotic division ~58- are listed in Tables 13 and 14, 'the statistical analysis of these data is shown in Table 150 The analysis indicated that colchicine treatment significantly decreased the number of mitotically dividing nuclei, the experimental F ratio for the combined treatment effects ~ 260753/50172 = 5.18 (see Table 15); theoretical F[[Z 00~ = 2011. Further, by subdividing the combined treatment sum of squares into the factors contributing to the treatment effects (time, concen­tration, and interactionl ), the following experimental F ratios were obtained~ Theoretical Factor F Ratio F Ratio (p-.05) Concentrat ion 58.823/5.172 := 11.37 2.48 Time 14.933/5.172 = 2.89 2.72 Interaction 19 0 018/5.172 = 3.68 1.88 All experimental F ratios for the 3 factors in this study exceeded their respective theoretical F ratios at the 5 per cent probability level (ratios for the concentration and interaction factors were highly significant, (P<oOl). The effects of colchicine, in concentrations from 0 0 1 to 0 0 8 per cent, on nuclear division in Allium roots are listed in Table 130 The results indicate that when time of • exposure was eliminated as a factor, all colchioine concen-trations employed in this study had an inhibitory effect on lInteraction refers to the manner in which the effects of the other two factors (time and concentration) interact with one another. Time (Hrs .. ) MNDN+ % GMt % Control 3 6 9 Table 13 Effects of Colchicine on Nuclear Division in Allium Roots Main Groups--Concentrations 0 .. 1% 0 .. 2% 0.4% 12 3 6 9 12 3 6 9 12 3 6 9 3.9 3 .. 6 405 400 205 2,,6 2.6 206 3 .. 1 3 .. 6 2.4 4.1 1.9 2.7 2 .. 1 4.00 2.58 +Mean number of dividing nuclei. T Group means. 3.30 1.90 --...... ----... - ----.--.... -- 008% 12 3 6 00 9 2.4 5.3 2053 9 1.5 12 00 9 i \n -.0 ti Colchicine % .. HNDN" % GMt % Control ~- -= == 3.9 3.6 4.5 4.00 Table 14 Effects of Colchidne on Nuclear Division in Allium Roots Main Groups-=Time 3 Hrso 6 Hrs. 9 Hrs. <>= 00 1 002 0.4 0.8 0.1 0.2 0.4 0.8 0.1 0.2 0.4 400 2.5 3.1 1.9 2.4 2.6 3.6 2.7 5.3 2.6 2.4 2,,1 2.48 3.55 2.15 +Mean number of dividing nuclei. TGroup means. 12 Hrs. 0.8 0.1 0.2 0.4 1.5 2.6 4.1 0.9 2.13 0.8 0.9 I I I 0' o I Table 15 Analysis of Variance+ of Colchicine Effects on Nuclear Division in Allium Roots Adjustment for Mean 9308.00 Mean Square Nature of Variation Degrees of Sum of Freedom Squares Between Treatments 19 508.31 26.753 Different levels of concentration 4 235.29 58.823 Different levels of time 3 44.80 14.933 Interaction 12 228.22 19.018 Error 80 413.73 5.172 Total 99 922.04 ---, ~ ~-... ---.... --..... --...... --...... -- +Transformation of data to angles. I 0" ' I--' i -62- the total number of dividing nuclei as represented by the group mean values (see row labeled GM) when compared with the group mean obtained for the control. Except for the 0 0 2 per cent concentration, the group mean values were signifi­cantly different than the control value. Except for the 12 hro exposure to the 002 per cent colchicine concentration, the mean number of dividing nuclei (see row labeled MNDN) for exposure times of 9 and 12 hrs. decreased as the con­centration of colchicine was increased. A flStudent ll t test performed between control and test means for 9 and 12 hrso exposure to 0 0 4 and 008 per cent colchicine indi­cated a significant reduction in the number of dividing nuclei" Also, for colchicine concentrations of 0.4 and 0 0 8 per cent, the individual mean values decreased as the time of exposure was increased from 3 to 12 hrs. There was little effect produced on the mean number of dividing nuclei (MNDN') either by increasing the colchicine concentra­tion for the 3 and 6 hro exposure times or by increas-ing the time of exposure at colchicine concentrations of 0 0 1 and 0 0 2 per cento The effects of colchicine on nuclear division arranged with time as the major variable are listed in Table 14. Individual values for the mean number of dividing nuclei (MNDN) indicate more clearly than in the previous table, that for exposure times of 9 and 12 hrs", there was a decrease in the number or dividing nuclei as the concen­tration or colchicine was increased. The relatively close values ror the 3 and 6 hrso exposure times with an increase in colchicine concentrations may also be seen. It is inter­esting to note the group means (GM) in Table 14, Which were calculated to eliminate the errects or the colchicine con­centrations as a ractoro A comparison or the dirrerences between group means ror the 6 hr. time exposure and any or the other times by the "Student" t test indicated that the dirrerences were signiricanto The high mean value ror the 6 hro exposure time (3~55 per cent) was primarily due to the high individual value or 503 per cent, obtained ror the 0 0 8 per cent colchicine concentration (also see Figure 8, 6 hro exposure time, page 65). Since this value was completely dirrerent rram other results in this entire experiment, and since it railed to rollow the general trend established for the other concentrations and exposure times, it seemed important to perrorm an extended study or the nuclear division rate in roots exposed to an 0.8 per cent concentration or colchicine ror a 6 hr. period or timeo The results obtained by studying an additional 20 roots rrom 20 Allium bulbs showed the mean number or mitotic div­iding nuclei to be 302 per cent; therefore, the 5.3 per cent value obtained in the original experiment was assumed to be aberrant and ror rurther graphical purposes (Figure 8) the -64- 3.2 per oent value was used. Figure a shows graphioally the effeots of oolohicine on nuolear md.totl0 division in the Allium roots for the four exposure times studied; 3, 6, 9, and 12 hrs. The conoentration of oolohioine (per oent) is plotted along the absoissa and the number of dividing nuolei, expressed as per cent, is plotted along the ordinate. In addition to showing values for the total number of dividing nuolei (solid linesl from Tables 13, 14, and 15, this figure shows the followingl 1. the total number of abnor.mal metaphases (dashed lines), in whioh typioal ohromosome o-pair types are present, ~, figure-as, foroeps, oruoiform; and 2. the total number of anaphases and telophases observed (dotted lines). It may be seen from Figure a that, in gen­eral, the total number of dividing nuolei deoreases with inoreasing oonoentrations of oolohioine. The most marked deorease in mitotio nuloear division may be seen at the 12 hr. exposure time. Thus, only 0.9 per o~nt dividing nuolei were observed for both the 0.4 and 0.8 per oent col­chioine oonoentrations. The deorease in the total number of dividing nuolei at the oonoentrations of oolohioine stud­ied, indioate that the colohioine employed was inhibitory to the overall··m.unl:ler of dIviding nuolei at and above oon­oentrations of 0.1 per oent. Colohioine, in the oonoentra­tions employed produced typioal c-mitotic effeots. At the H ;..u ....:1- tt.,O..,.:l (') i"J r' >--.' Z d) ~r:; (.) lcr1 CJ [I" Z I.., ~HCD ::':d:::tIJ.. ~ t-",J,,-/ h c::1 I-j (xJ ,....:1""' 5 4 J 2 1 o~ .- 5 ,~-.~, t~.), ;,.~0 J -- Z ID &~_, u C"Cl Z k §Ci:e 2 :z H'-" ::> H >=l 1 o 0 Tot.a~, n'l!r.be!" of dividing nuclei. a-- -0 Total nu.rnber of abnormal meta phases. 0-' •. -. ¢ Total mt!"~ber of anaphases and telophases C!ombined. J HOURS 6 HOURS . ...0- - - --"--- - --<>- - - ---<) . ,...- ,...--. ..",."... .......... ~ ~ ..... '"' ..... ...................... (1)- ...... » /' ....,A..... , .",... .......... .......... / "-0- _ - _-0 ...... y./ o~ ,...,- ". ·If····· .. . ... . .< I>- .. ......... -... A "" ¢ It "'see page 63 for explana:ion of t:,is 9 H-JURS '. _ --0- - __ --0-­, .. A ,,/ "" /" "- ,,/ " '. '. " " ------0 .. -0- o .... ----":";·~. p ........... .,. ......... • .. ·t· ..... '" ·It • ., ,,/ "" ok----JP· .... .. " '" .¢ . ........... ...?i >=: ........- :". -;-: :-:-.-=" 0.1 0.2 0.4 Cv·". "v' LrHI_r.L~l "~.'f-i,' (F er ~"eY".'l t ) 0.8 0.1 0.2 0.4 COLCHICINE (Per cent) 0.8 rig. 8. Effects of Colchicine on Nucl~.ar Livision in Allium Roots. I 0"­\ Jl. a -66- 3 and 6 hr. exposure times, the number of abnormal metaphases was observed to increase with each increase in colchicine concentration. Maximum effects were observed to occur at the 3 hr. exposure to 0.8 per cent colchicine and at the 6 hr. exposure to 0.2 per cent colchicine. At the 3 hr. exposure time to 0.8 per cent colchicine, 1.4 per cent of the total 2.5 per cent of dividing nuclei showed abnormal metaphases (56 per cent of the total number of dividing nuclei observed). In addition, it may be seen that the combined number of anaphases and telophases was virtually eliminated at any of the 4 colchicine concentrations em­ployed and for any exposure time studied (the anaphases and telophases were completely absent at the 12 hr. exposure time to concentrations stronger than 0.2 per cent colchicine). 4. Discussion The data presented indicate that colchicine is cap­able of modifying nuclear mitotic activity by producing typical c-mitotic changes in the chromosomes of Allium ceEa roots. The ccncentrations of colchicine employed in this study (0.1 to 0.8 per cent) not only inhibited the overall ntunber of dividing nuclei, but also induced a large number of abnormal metaphases to occur, character­ized by shortened, thickened chromosomes in distinct c-mi­totic appearance. For example, the usual coiling of cleft -67- chromosomes was markedly reduced with a resultant diminu­tion of the number of turns for each segment; this effect gave the chromosomes a figure-8 and forceps-type of appear­ance. When the cleft chromosomes had completely uncoiled but not yet separated, a number of X-shaped (cruciform) c­pairs were observed that were held only at their centro-meres. Because there was a complete absence of any semblence of an equatorial plate during the metaphases, and because of the rapid decline in the number of observed anaphases and telophases whenever colchicine was employed, success­ful inhibition of the spindle apparatus was assumed. In addition, in a number of instances chromosomes in c-ana­phase were observed to have separated at the centromeres from their metaphase X-shapes and thus, each chromosome was lying beside its duplicate, giving the appearance of a pair of "skis". At this point, the usual diploid (2n = 16) chro­mosome complement present in Allium cells had doubled and formed a true polyploid cell (2n = 32). Since the spindle apparatus had been inactivated and two new daughter cells did not form, a completion of a chromosome mitosis without a nuclear or cellular mitosis had been effected. The highly significant value (3.68) obtained for the interaction factor possibly explains why the time factor just exceeded the 5 per cent significance level. This indl- -68- cat ed, therefore, that the effects produced by different concentrations of colchicine employed to modify nuclear mitotic division were dependent upon the leng.th of time the roots were exposed to the agent. Therefore, in any future study of this nature using colchicine, not only must the concentration of this agent be taken into account, but also the exposure time must be considered. Colchicine-induced modification of nuclear mitotic division in the Allium bulbs was compared with the effects observed for other agents (Veriloid and protoveratrine A) to determine the activity and relative potency of these new agents on nuclear mitotic division. -69~ B. The Effects of Veriloid on Nuclear Division 1. Introduction Although veratrine has been reported to be effective in producing chromosomal changes resulting in polyploidy in Allium cepa L. (Witkus and Berger, 1944), the litera­ture contains few reports involving the application of veratrine or related agents to the inducement of polyploidy in plants. Sonnenschein (1941), following an investiga­tion of the effects of several alkaloids on polyploid production in soybeans, reported successful pOlyploid results using a 0.2 per cent solution of veratrine. Also, Martinez (1949) found that veratrine (0.1 per cent) modi­fied the growth of wheat seedlings during a 4 to 9 day test period. No reports of the effects of other agents structurally related to the alkaloidal constituents in veratrine (cevadine and veratridine) were found. Inasmuch as the numerous alkaloidal constituents present in plants of the Veratrum genus possess a basic steroidal configura­tion which is similar to the veratrine alkaloids found in sadadilla seeds (Schoenocaulon officinale A. Gray; the taYonomiB~ A. J. Retzius, prefers to place sabadilla in the Veratrum genus, Veratrum sabadilla), it seemed impor­tant to determine the effects of an agent on nuclear mitotic division which was representative of the alkaloidal consti. -70- tuents present 'in many plants of the Veratrum genus. Veriloid was chosen for these studies, since this agent represents a standarized mixture of alkaloids present in Veratrum vir ide L. 2.. Methods A population sample of 100 Allium bulbs was randomly divided into 5 main groups of 20 bulbs each. Each of the main groups was further randomized and subdivided into 4 subgroups of 5 bulbs each.. The roots on the bulbs in 4 'of the main groups were treated with one of the following oonoentrations of Veriloidl : 0.1, 0.2, 0.4, or 0.8 per oent. The remaining main group served as a control. The roots of the 4 subgroups were exposed to the requisite Veriloid solution for a predete~ined period of time, i.e., 3~6J 9, or 12 hrs. The Veriloid as supplied from the manufacturer was soluble in concentrations of dilute aqueous solutions of aoids such as acetic acld, phosphoric acid, etc. In order to dissolve the Veriloid, a minimum amount of Diluted Acetic Aoid N.F. (l.~ml .. /IOO ml. of solution) was employed. This amount' of acid (equivalent to 0.084 gms. of C2H 4 02 ) was found, by examination of the cells, to be non-injurious lconcentrations equivalent to Reference S':;andard Alkavervir (Veriloid), Riker Laboratories, Los Angeles, Calif. -71- to the root tissues o Fresh solutions of the agent in acidi­fied tap water were used throughout the experiment, and all procedures were conducted at a solution temperature of 300 e. !5°e. A statistical comparison of the effects produced on nuclear mitotic division by different concentrations of Veriloid and by different durations of exposure to the agent was performed by means of the analysis of variance technic using a completely randomized design with a factor-ial arrangement of the treatments a 30 Results The effects of different concentrations of Veriloid and different times of exposure on nuclear mitotic division are listed in Tables 16 and 17; the statistical analysis of these data is shown in Table 18. The analysis indicated that Veriloid treatment significantly decreased the number of mitot-ically dividing nuclei, the experimental F ratio for the combined treatment effects ~ 12 e 595/5.128 = 20 46 (see Table 18); theo­retical F~~ .o~= 2.11. Further, by subdividing the treatment sum of squares into the factors contributing to the treatment effects (time, c~ncentration, and interactionl ), the follow-lFor explanation of the term interaction, see the footnote on page 580 Tim.:! (Hrs.) t1NDN + % GM+ % Control 3 6 9 12 5.1 4.9 5.5 509 Table 16 Effects of Veriloid on Nuclear Division in Allimll H.oots Hain Groups~-Concentrations 0.1(" 0.2% 0.4% 3 6 9 12 3 6 9 12 3 6 4.5 3.4 7.6 g.9 5.7 5,,5 7"g 7.3 7,,1 5.9 9 7.3 5.35 6elO 605g 6.80 +Alkavervir (Veriloid) Reference Standard (see page 70). +Mean number of dividing nuclei • .:t'Group means. 0 .. 8% 12 3 6 6.9 6.6 5 .. 9 6070 9 12 7 .. 6 o/ . ...(.. ! ! -J I'\) I Veriloid+ % MNDN1- % ~ % Control -~ -- -- -- 5 .. 1 4 .. 9 5.5 5.9 5.35 Table 17 Effects of Veriloid on Nuclear Division in Allium Roots Main Groups--Time 3 Hrs. 6 Hrs. 9 Hrs .. 0.1 0.2 004 0.8 0.1 0.2 0.4 0.8 0 .. 1 0.2 0.4 405 5 .. 7 7.1 6.6 3.4 5 .. 5 509 5.5 7 .. 6 7.8 7 .. 3 5.98 5 .. 08 7058 +Alkavervir (Veriloid) Reference Standard (see page 70). tMean number of dividing nuclei. * Group means. 0 .. 8 0.1 7.6 8.9 12 Hrs. 0.2 00 4 7.3 6.9 7045 0.8 6.7 i -J W ~ Table 18 Analysis of Variance+ of Veriloid Effects on Nuclear Division in Allium Roots Adjustment for Mean 20899.907 Mean Square Nature of Variation Degrees Sum of of Freedom Squares Between Treatments 19 2390309 12.595 Different levels of concentration 4 42.854 Different levels of time 3 1130166 Interaction 12 83.289 , Error 80 410.198 5.128 Total, 99 649.507 L-- --...... ---.... --..... ~- --- '---- +Transformation of data to angles. 10.714 37.722 6.941 I -.J +:­I -75- ing experimental F ratios were obtained: ,Exp (I Theoretical Factor F Ratio F Ratio {P= "O!2l Concentration 100714/5,,128 • 2009 2 .. 48 Time 37,,722/5,,128 -- 7036 2.72 Interaction 60941/50128 = 1 .. 35 1.88 Only the experimental F ratio for the time factor in this study exceeded the theoretical F ratio at the 5 per cent probability level (it also exceeded the 1 per cent level, F ~ 4 (04); therefore, the length of time in which roots were exposed to the Veriloid appeared to be of greater importance to nuclear mitotic division than were the con­centrations employed" The effects of Veriloid, in concentrations equivalent to Reference standard Alkavervir (Veriloid) from 0.1 to 0,,8 per cent, on nuclear division in Allium roots are , listed in Table 16" The results from the analysis of variance indicate that the Veriloid concentrations, eliminat-ing time as a factor, employed in this study had no signifi-cant effect on the total number of dividing nuclei as rep­resented by the group means (see row labeled GM) when com­pared with values obtained for the control. The effects of Veriloid on nuclear division, arranged with time as the main groups, are listed in Table 17. For each time tested, the individual values for the mean number of dividing nuclei (see row labeled MNDN) indicate a gen­eral increase in the mean values as the Veriloid concen­trations were raised. As shown by the experimental F value for the time factor from the analysis of variance calcula­tion (see page 75), a highly significant figure (7.36) resulted o The figures in the row entitled Group Means (GM) in Table 18, so calculated to eliminate the effects of the Veriloid concentrations as a factor, are the mean values for each time tested c A comparison between the group means for the 3 hr .. and 12 hr" exposure times, 5 .. 98 and 7045 per cent, respectively, revealed that the differences were significant (p 005) when examined by a "Student" t test.. Further, the most marked increase in the total num­ber of dividing nuclei occurred between the exposure times of 6 and 9 brs .. , and differences between the group means for these 2 exposure times were highly significant (p .01). Figure 9 shows graphically the effects of Veriloid on the nuclear mitotic division in the Allium roots for the 4 exposure times studied, 3, 6, 9, and 12 hrs. The concen­tration of Veriloid in per cent (equivalent to Reference Standard Alkavervir) is plotted along the abscissa and the number of dividing nuclei expressed as per cent, is listed along the ordinate., In addition to showing values for the total number of dividing nuclei (solid lines) from Tables 16, 17, and 18, this figure shows the following~ 9 8 H 7 i:1l rx..oHo"+--->'" 6 o~@ ~ () .5 \:::d 0 fIj;z; j:..., -, H Q) 4 ..... 00... ~H'~- '" 3 Q 2 1 0 9 8 H 7 i:1l .....:l-f: r.. U +> 6 o~~ ~ 0 .5 j::;:ld 8 2 j:..., 4 ~HQ) !::')oo... ;Z; H"""'" 3 S 2 1 0 o 0 Total number of dividing nUclei. 0----0 Total number of abnormal metaphases. o· .~ .... «) Total number of anaphases and telop'bases combined. 3 HOURS • .0' •..•••.•••• ·0 .......•.. , .. , "0' ••..•.•.... "0' ,.' .. " .. ,.' 9 HDur~ .. ' ..•.•• ' • .0 .••••••••. 0· . . . .. '0' • ••••••• .0 --- -- .-.-.---- 0.1 0.2 0.4 0.8 VERILOID (Per cent) 6 HOURS .0 .•.•••• " " "0 ' .. "'0" .•.•• ' •.• ' . o' .. ' . o ~ == =b= :;p= =1?=- =4 .0. . . '0' . ~ •....... 0·· '" ...... 0 -- 0'=-"";'--""--- ::r .-D-- -- ------0 I 0.1 0.2 0.4 0.8 VERILOID (Per cent) Fig. 9. Effects of Veriloid on Nuclear Division in Allium Roots. I .~ -.J I -78- the total number of abnormal metaphases (dashed lines) in which abnormal chromosome types were present, ~, thiokened and contraoted chromosomes, lack of equatorial orientation of chromosome pairs, etc o , and the figure shows the total number of anaphases and telophases observed (dotted lines)o It may be seen from Figure 9 that, in general, the total number of dividing nuclei remains relatively unaffected by increased concentrations of Veriloid (a slight but insignificant increase may be the overall effeot). This effect was also pointed out from the analysis of variance calculation (Table 18) and from examination of the concen­tration effects of the Veriloid given in Table 16. The total number of dividing nuclei remained relatively un­changed and the number of abnormal metaphases (dashed lines) increased slightly when roots were subjected to the influences of the Veriloid o The total number of anaphases and telophases (dotted lines) was not significantly altered during the entire study from mean values obtained for the controls 0 40 Discussion The data presented indicate that alkaloidal agents present in Veratrum viride Aito have an effect on nuclear mitotic division in roots of Allium cepa L. The Veriloid employed in this study was shown to have a -79- marked influence on the total number of mitotic dividing nuclei (Table 18 and page 75)0 Indeed, the concentrations of Veriloid used (001 to 008 per cent) were relatively unimportant since the number of dividing nuclei remained very close to the value obtained for the control (Table 16). At the same time, the length of time to which the Allium roots were exposed to the agent proved to be extremely im­portant o Exposure of the Veriloid solutions to the Allium roots for periods of 9 to 12 hrs., irrespective of the concentrations used, increased the number of dividing nuc­lie by a highly significant magnitude (Table 17 and page 75)" Therefore, although no appreciable changesocourred in the total numbers of metaphases, anaphases, or telophases observed over that noted for the controls (Figure 9), Veriloid, nevertheless, markedly modified nuclear mitotic division. Chromosomal aberrations were frequently observed, ~, contracted and apparent adhesive properties of the cleft chromosomes at metaphase resulting in a lack of diverged chromosome ends (X-shapes)" Further, apparently as a result of these "sticky" chromosomes, many anaphase bridges were observed especially after extended exposure periods to Veriloid" Occasionally a few cells were ob­served in which the normal 2n = 16 Allium chromosome com­plement had apparently been doubled or quadrupled (2n = 32, 2n = 64)0 These findings were in agreement with the -80- results reported by Witkus and Berger in 1944 with veratrine as the experimental agent in the same species of plant as that used in this study. They found that veratrine sulfate was effective in producing polyploidy in three ways: 1. by oomplete inhibition of spindle formation; 2. by fusion of daughter nuclei after incomplete anaphase separation; and 3. by failure of anaphase .separation due to "sticky" chromo­some bridges. The results observed for the Veriloid (from Veratrum viride Ait.) in this study showed effects on nuclear mitotic activity in agreement with those reported previously for veratrine (from Schoenocaulon officinale A. Gray). There­fore, it appears that alkaloids in Veratrum vir ide struct­urally similar to those found in veratrine have effects on nuclear mitotic diviSion in root tips of Allium cepa L. which resemble the effects produced by veratrine. -81- o. The Effects of Protoveratrine A on Nuolear Division 10 Introduction The reoent attention given to the extracts obtained from Veratrum for the treatment of hypertensive disorders has resulted in extensive investigations attempting to isolate, purify, and oharaoterize many of the Veratrum alkaloids. Protoveratrine, first isolated by Salzberger in 1890, is one of the hypotensively aotive alkaloidal prinoiples. For many years protoveratrine was believed to be a pure alkaloid; however, Klobs et ale (1952) and Nash and Brooker (1953) reported the isolation of 2 orystalline fraotions whioh they obtained from "protoveratrine" found in Veratrum viride Ait. and Veratrum album L., respeotively. These fractions are now recognized as Protoveratrine A (protoveratrine) and protoveratrine B (neoprotoveratrine, veratetrine, alkaloid VB). Because the protoveratrines are known to be present in Veriloid (discussed in detail in the previous section), and in order to test the effects on nuclear division of an alkaloid found in another Veratrum genus, Veratrum album, protoveratrine Al was chosen for these studies. lSuPPlied by Riker'Laboratories, Los Angeles, Calif. and Pitman-Moore Company, Indianapolis, Ind. See also foot­note on page 8. ~82- 2. Methods In the course of investigating the effects of proto­veratrine A on nuclear mitotic division, it became apparent that the changes produced were very nearly the same as those observed for Veriloid. For this reason, the experiments in this section are not as extensive as that reported in the previous section. A population sample of 25 Allium bulbs was used in this study. The sample was randomized into 5 groups of 5 bulbs each. The groups represented 4 times of exposure (3, 6, 9, and 12 hrs.) to an acidified 0.025 per cent solution of protoveratrine A (see page 70) and a control group containing no protoveratrine. At the end of each test time, roots were killed, cells were observed, and the total number of cells in all stages of nuclear mitotic division was calculated. The ratio of this number to the total number of dividing cells present was expressed in per cent value. A comparison of the effects produced on nuclear mitotic division in each of the 5 groups was per­formed by means of the analysis of variance technic. 3. Results The results obtained showing the effects of dif­ferent exposure times of protoveratrine A on nuclear division are listed in Table 190 The mean values suggest some increase in the number of dividing nuclei from that observed for the controlo The experimental ratio from Table 20 was F = 2.603/40771 = 0 0 546, from the table of theoretical F values, F ~~ .051 = 2.87. Since the experimental F value did not ex­ceed the F value obtained from the table at the .05 level of probability the differences in nuclear division were not signif­icant for the 3, 6, 9, and 12 hr. exposure times tested. Figure 10 shows graphically the effects of protovera­trine A on nuclear mitotic division in the Allium roots for the exposure times tested. The lengths of exposure (hours) to the protoveratrine A are plotted along the ab-scissa and the number of dividing nuclei, experessed as a per cent value, is plotted along the ordinate. The solid, dashed, and dotted lines represent the total number of dividing nuclei, the total number of abnormal metaphases (see page 78), and the total number of anaphases and telo-phases, respectively. It may be seen fram Figure 10 that the total number of dividing nuclei remained relatively unaltered by different times of exposure of the roots to the protoveratrine Ao A slight but insignificant increase over that observed for the control value may be the over­all effect produced; this may also be seen for the mean values listed in Table 19. The number of abnormal meta-phases increased considerably for different times of ex­posure to the protoveratrine A. At the 6 hro exposure Table 19 .Gstil!late. of l'fuc! , .. 'J,r Dl vi,s] on 'reans for T.ffcrent T;:,:ws of K(no::mre 1,0 Protoveratr~;,() •• 'rimes Exposure Mean ol /::J of Dividing Standard to :'rotoveratrine , NucLei Error L Control 508 3 ~{rso 6.6 6 hrs& 706 ~ .69/5 = 9 fir;;. 6.7 12 Hrso 6.9 Analysis of Varia:1ce+ of Protoveratrine .'. Effects on !~uclGar Dlvision in nH:lt'1l :too!~:; ;,d justment for Hean 5633.2-0 De§Fees NatlJre of Variation Sum of Freedom Squares Between exposure times 4 10.41 Error 20 95.42 Total 24 105083 +Transformation of data to angles o .37 Mean Square 2.603 46771 H ~ H o ~ 0"-" ;2;....> ~ § H 0 ."., H $.., P Q) ,,-<f:::­o ffi Efl ~ Z o 0 Total number of dividing nuclei. 0---0 Total number of abnormal IT'.etaphases. 0······ .. 0() Total number of anaphases and telophases combined. 8 7 6 5 4 3 .Q ••••• .. .. .. .. . '0 . . .. <I" • 0 .. " .. .. .. .. • .. .. .. .. .. " "0'· .. .. .. .. .. .. .. .. .. .. .. .. 2 .. 1 __ .....-. ---0--........ ......... _ --- -.0--- -_ ---0--_____ ---- 0 o 11 ......... -- I I 3 6 9 12 EXPOSURE TIXE (Hours) Fig. 10. Effects of Protoveratrine A on Nuclear Division in Allium Roots. I CP \.J1. 8 ~86- time to the protoveratrine A, 1 per cent of the observed nuclei showed abnormal metaphases in the same manner seen in the Veriloid study. Therefore, treatment of the Allium roots with protoveratrine A produced mitotic nuolear ohanges although the number of mitotic dividing nuclei remained relatively unaffected. 4. Discussion The data presented indicate that protoveratrine A~ a purified, crystalline alkaloid found in Veratrum album L. and Veratrum viride Ait. is capable of modifying nuolear· mitotic division in roots of Allium cepa. Although the greatest value attained for the number of abnormal metaphases was only 1 per cent at the 6 hr. ex­posure time, such a value would be highly significant when compared with those obtained with non-treated roots. Sel­dom does the number of observed abnormal metaphases ever make up even 0.04 per cent (less than 1 in 2000) of the total number of nuclei observed in untreated roots. There­fore, assuming there was 0.0$ per cent of abnormal meta­phases in untreated Allium roots, exposure to the 0.02$ per cent solution of protoveratrine A for a period of 6 hrs. caused a 20-fold increase in the number of observed dividing nuclei. Chromosomal aberrations were frequently observed which assumed appearances fairly typical of Veratrum- = 8''1'' = induced changes, i"e"l> contracted and "sticky" chromosomes resulting in closely adhering arms of cleft chromosomes, and many anaphase bridges o The changes observed using protoveratrine A as the experimental agent were indistin­guishable from the changes seen in the previously reported Veriloid studyo -88 Vlo GENERAL DISCUSSION The data presented indicate that 2-chloroethanol treatment of Allium bulbs did not interfere witb nuclear mitotic division although the agent was shown to stimulate the production of new roots (see section III). This observation is in agreement with the com­meroial application of this agent for breaking dormancy and stimulating growth in other plant genera (Denny, 1938; Townsend, 1941; and Johnson, 1946)0 In the experiments relating root length and nuclear mitotic division, acceptable limits to the length of the roots were determined in which the mitotic nuclear division was relatively constant o The fact that short roots had fewer dividing nuclei than long roots and the fact that reoently sprouted roots elongate more rapidly than older roots, indicate that plant growth involves two independently functioning processes (cell division and cell elongation) and is in agreement with previous reports on this subject (HeynJ 1940). The results obtained in the study of the influence of chemical treatment (2-chloroethanol), root length, and environmental alterations (light and temperature) on the number of mitotic dividing nuclei were used to eliminate these variables in the ensuing colchicine and Veratrum studies. -89- Temperature, one of the most variable of environmental factors, plays an important role in plant growth. Exposure of plants to cold and hot temperatures has been shown to double the number of plant chromosomes {Dorsey, 1936; and Beasley, 1940)0 Dermen (1938, 1940) compared the effect­iveness of temperature on the production of polyploidy with that induced by colchicine treatment o As a result of Der­men's and other work, it is now estabished that temperature, although occasionally effective, is not as efficient in the production of polyploidy as colchicine. The data obtained for colchicine, Veriloid, and proto­veratrine A are summarized in Table 210 It may be seen from the table that all three agents produced polyploidy in Allium roots, although, in general, the effects were much more pronounced after colchicine treatment o Further­more, a comparison of the effects produced by the Veratrum derivatives on nUclear mitotic division with those observed for colchicine revealed many interesting similarities and differences 0 The equatorial plate formed during prophase and along Which the chromosomes align themselves during metaphase was completely absent in the colchicine-treated root tips, whereas apprOXimately 60 per cent of all meta­phases observed in the Veratrum-treated root tips showed ohromosomes in regular alignment in the equatorial region. This was one of the major differences between the Veratrum Table 21 S~~ry of t~e Effects of Differe~t Liliaceous Alkaloids on Nuclear J'.itotir:: Iivision in Roots of Alli'.lm ~* Colc:licine Concentrations Employed (Per cent) C.l, 0.2, O.lh 0.3 Times of Exposure (Hrs.) '{ -,p 6, 9. 12 Polyploid pro"j ~,e ~~, Equatorial Plates a'bse,~)'~ Chromosone Characteristics Thickened ',;i th rrlin i.rri\LI1 bdsting pro;":,',, r.ent Fig~)re=8s and for~ep5 prc:r~'i :V:"~1~., Cruci fom: pr:;rri'i :J.e:1t Anaphase IIski.!! pairs p.Y"01'.' ":e.·'.t "St,iel{yll a!l.aphase bridges absent Total Dividing Nuclei CCDcentration effects decreased ~ Time effects decreased ~ Total Abnom.al l".etaphases COD;c;entration effects increased l' Time effects deet'c<5ed ~ Total .A.naphases and Telophases Cor,,:::ent.ration effects decrea3ed ~ Tirr.e effects unaltered ~ * ~ == significant increase over co~trol value (p<. 0.5). ~ := significant decrease under control value (p<. 05). e = not significantly different from contr·jl value (p >.05). - = not determined for this agent. Vel'i.loid 0.1, 0.2. 0.4, 0.8 .'"J p 6, 9, 12 preserlt. present pro Jr. 1,:, e r-J t. presenL=-lIst icky'; t.yp absent preser,t==angi.ed prominent increased ~ increased l' increased l' increased t increased 1- increased t Protoveratrine A j 0.025 i ~ r G 12 I ...;t b t /' presen~:. preS6:1t Er-G~i~"'..e. '~.t" l ~.., preser:t=-lI s-1.(cky" typ! absent presen~=-.3)'P"j P.r1 prorr.inen~~ --- increased f-) --- increased l' --- increased. ~ u -D o i -91-= and colchicine treatments and suggested that the deriva-tives of the former do not act specifically on the mitotic apparatus per ~, but possibly by some modification of cytoplasmic function. It was impossible to differentiate between the two Veratrum agents employed solely on the basis of the observable chromosome characteristics. How­ever, except for thickened, rather poorly twisted arms of cleft chromosomes which were noted with all three agents, the chromosomes of colchicine-treated cells differed markedly from the chromosomes in cells treated with Veri-loid or protoveratrine A. Thus, the colchicine effects (figure-Bs, forceps, cruciform, and "ski" pairs) were either absent, or if present, asstnned very different forms ("sticky" or angled chromosomes) than those observed in Veratrum­treated roots. The phenomenon of anaphase bridges, whioh was very prominent in the Veriloid- and protoveratrine A­treated roots, was never noted in the colchicine study. A comparison of the effects produced by the Liliac­eous alkaloids on the total number of mitotic dividing nuolei in Allium roots, as well as on various stages of the mitotic process (metaphase, anaphase, and telophase), brought out other major differences. It may be seen from the table that colchicine treatment decreased signif­ioantly (p < 005) the overall ntnnber of dividing nuclei in cells irrespective of concentration or time of exposure. In marked contrast, Veriloid and protoveratrine A treat­ment consistently inoreased the number of dividing nuclei, provided, however, that the time of exposure for Veriloid was adequate. Because colchicine effectively blocks nuclear division at approximately the halfway point through the mitotic cycle, but has no effect on stopping initial nucl~ar mitotic division, the overall number of divid-ing nuclei would be expected to increase at any given time by virtue of a "piling up" effect. Instead, the overall number of dividing nuclei decreased significantly. Such a finding probably indicates that the colohicine conc­entrations employed (0.1 to 0 0 8 per cent) were too high to effect an optimum mitotic effect. The total number of abnormal metaphases was significantly increased by all the Liliaceous alkaloids, in some instances as much as 20-fold. The most marked differences between colchicine and the Veratrum agents were noted with the anaphase and telophase stages o Whereas colchicine treatment consist­ently reduced the number of observable anaphases and telophases (often to the point where none were to be seen after colchicine treatment), Veriloid or protoveratrine A treatment was just the opposite; an increase, in general Significant, was regularly notedc Thus, colchicine, by inhibiting the mitotic cycle at metaphase prevented any anaphases or telophases from forming and caused cells =93- already in these two stages to progress, divide, and assume a resting state as new daughter cells. On the other hand, the veratrums do not act directly on the mitotic apparatus and, consequently, many anaphases and telophases were to be observed after treatment with any concentration of these agents. Colchicine and closely related structural compounds are the only chemical agents which have been shown to act specifically on the mitotio apparatus in living cells. The ability of oolohicine, in a wide range of concentrations to act selectively to abolish formation of the continuous (cell plate) and disoontinuous (chromosomal) spindle fibers in both plant and animal cells, provides a clearer inSight into the mechanisms involved in mitotic processes and the roles they play in oell divisionQ This study has shown that alkaloidal agents (mixtures or crystalline) from Veratrum viride Ait. and/or Veratrum album Lc having structural similarities to alkaloids obtained from sabadilla seeds (Schoenocaulon officinale A. Gray), may produce polyploidy in roots of Allium oepa by an action similar to that of veratrine (Witkus and Berger, 1944) but which is markedly different and appar­ently inferior to the mode of action seen in colchicine­treated roots o VII.. SUMMARY AND CONCLUSIONS This study represents an investigation and oomparison of the effeots of various Veratrum alkaloids on nuolear mitotio division in an effort to determine 1. whether some purified alkaloidal mixtures or orystalline alka­loids possess the ability to modify the oyole of nuolear and oellular division, and 20 to determine how suoh aotivity oompared in kind and degree to that already well established for oolchicine. In addition, prelim­inary studies were conducted to determine what effect a number of other physical and chemical factors had on the nucleru: mitotic division being studied in order to eliminate their influences as experimental variables. Onion bulbs, Allium ce~a varo White Portugal, were used as the experimental plante Roots were sprouted on the bulbs using tap water and experiments were conducted under constant temperature (30 oc.+0.5°c.) conditions. Roots from 1.5 to 6.0 cm .. :i.n length were used for all observations of nuolear divisiono Microsoopio examin­ation of the cells was performed by a root tip smear method in which root tips were killed and rixed for 24 hra. or longer, softened to separate the individual cells, rehardened, stained, and observed through a microscope ~t a magnification of 4~.0 diameters.. All intact cells =95= observed in a predetermined area of a glass slide, either resting or in any stage of mitotic nuclear division were recorded and the per cent of cells in nuclear division was calculated o Suitable statistical technics were employed (chi-Square, linear regression, and analysis of variance) to analyze the experimental results and permit conclusions to be drawn therefrom Q A. The Effects of Preliminary Tests on Nuclear Division and Root Growth 1. 2-chloroethanol No significant differences were noted from oontrol values in nuclear division between times of exposure of 3 and 6 hrs. to a 2 per cent solution of 2-chloroethanol. Three hro bulb soaking failed to stim­ulate germination of a significantly greater number of bulbs that was produced by tap water alone; however, production of new roots was Significantly increased over results observed for tap-water controls. The data suggest that pre-soakjng periods for Allium bulbs were not harmful to nuolear mitotic division. Further, the agent appeared to be incapable of breaking bulb dormancy but was highly effective in increasing the germination rate of new roots from non-dormant bulbs (approximately 19 per cent over control values). 2. Root Length Although there was no significant difference in nuclear division between roots up to 7.5 cm. in length, very short roots (up to 1 0 0 cmo in length) were consist­ently low (2.4 per cent) in the number of dividing nuclei from that of the control (~-oO per cent) 0 The data suggest that short Allium roots up to 100 cmo long increase in length more from cell elongation that by meristematic mitotic production of new cells o To eliminate the influences of root length on nuclear division as an experimental variable, only roots from 1.5 to 6.0 cm. long were used for all observationso B. The Effects of Environment on Nuclear Division and Root Elongatio!!, 1. Effects of Light In a population sample of 40 Allium bulbs the number of mitotic dividing nuclei was not significantly changed for different times of day when observations were perfor.med on roots obtained every 3 hrso over a 24 hro period of timeo The effects of light, intermittent light, and darkness on nuclear division and root length over a 72 hr .. test period indicated that prolonged exposures to artificial light had a noticeable but non~significant inhibitory effect (p> ,,05') on mitotic nuclear division and a marked inhibitory effect on root length (P<.OOl). =9 The data suggest that to eliminate the influences of light as an experimental variable on the number of mitotic dividing nuclei, the Allium roots should be exposed to known intensities of the light which may be controlled at all times .. 2. Effects of Temperature Exposure of Allium roots to 7 different temper­ature gradients from 15 to 4.5°c~ in increments of ~c. for 24 hrs. revealed that the mean number of mitotic dividing nuclei exposed to 20, 25, 30, and 35°c. were not significantly different from the mean values obtained from roots exposed to the control temperature (300 C.), whereas means at temperature extremes of 15, 40, and 4~C. differed significantly (40 and 4.f C., P <.,01) from controls. The data suggest that rather wide temperature fluctuations may be permitted without producing undesirable alterations in mitotic nuclear activity" Temperature extremes, however, may have marked effects on mitotic division. C. The Effects of Llliaceous Alkaloids on Nuclear Division 1. Effects of COjchicine Examination of Allium bulbs treated with conc­entrations of colchicine of Ool~ 002, 0 0 4, or 0.8 per cent for predetermined periods of time of 3, 6, 9, or 12 brs. revealed that the time and concentration factors both caused significant changes in nuclear mitotic division from control values o Independent of the time factor, all colchicine concentrations had an inhibitory effect. When time was examined as the major variable, significant differences in nuclear mitotic activity were also observed. A.~alysis of the data also showed a significant interrela­tion between the effects produced by the time and conc­entration factors o Typical colchicine-mitotic (c-mitotic) effects were produced with all colchicine concentrations employed. The data presented indicate that colchicine is capable of modifying nuclear mitotic activity by producing typical c-mitotic changes in the chromosomes of Allium roots. Successful inhibition of the spindle apparatus may be achieved and polyploid cells (2n = 32) are prominent. When using colchicine experimentally, not only must the concentration be taken into account but also the exposure time must be considered. 2. Effects of Veriloid The effects produced by Veriloid, a standard­ized mixture of hypotensively active alkaloids present in Veratrum viride Aito were examined in Allium bulbs treated with acidified solutions which contained concentrations equivalent to 001, 0.2, 0 0 4, or 0.8 per cent of Reference Standard Alkavervir (Riker Laborator­ies) in fresh tap water for predetermined periods of time (3, 6, 9, or 12 hrs~)o The data suggest that =99= alkaloidal agents present in Veratrum viride have an effect on nuclear mitotio division in roots of Allium cepa L. Chromosomal aberrations were frequently observed although their appearance suggested a markedly different mode of action than that observed with colchicine. Polyploid cells may be noted and the effects on nuclear mitotic division are in agreement with those reported by other workers for veratrine, an alkaloidal mixture obtained from a different plant source but structurally similar to Veriloid. 3. E, ffects of Protoveratrine A The effects produced by protoveratrine A, a purified crystalline alkaloid present in Veratrum album L. were examined in Allium bulbs treated with a 0.025 per cent acidified solution in fresh tap water for pre­determined periods of time (3, 6, 9, or 12 brs.). The data suggest that protoveratrine A is capable of modify­ing nuclear mitotic division to produce polyploid cells in roots of A~liu~ cepa o The changes observed were indistinguishable from the changes reported in the Veriloid study. ~lOO" VIII. REFERENCES Arkin, A. and Colton, Ro He Tables for statisticians. N.Y., Barnes and Noble, Inc. 1950. Avery, G. G., Jr. and Johnson, Eo B.Hormones and horti­culture. N.Y., McGraw-Hill. 1947. 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