CiHANth m 2"?» a mama ASTEV‘ETY {35 THE aLKALGEEE CGEC‘E-E 9 $555.. 53:51:55. 51:15:55. SW55 5 ~ luv.“ 3';~‘ |‘.’ t‘ ‘, 6:; f“; . ' a' " 9"- o EOM‘ET'VM "2% 3:152;1:ag.::51zfi 5,? EV“? 5.5mmca‘ m. I‘ In it. Q "'3‘ "5’ M‘. c - a .gfia': 18¢ 15:1. I) Q 5.55“. “1:: m . 1-: W m :rxmmtfm imam—x. _. wflm ”$23832; 5'2: fine 80g?” :2? 535.. S. 535 5135.55 5553555355 .:.R u. g 2355253562 €52.55 Grumbe g. 3?”; THESlS LIBRARY Michigan State University ABSTRACT CHANGES IN THE BIOLOGICAL ACTIVITY OF THE ALKALOID CCLCHICINE, UNDER VARIED SIORACE CONDITIONS, AS MEASIRED 3? END CYECLCGICAL EFFECTS ON ggsgg SATIVUE Egg, ALASKA. by Edward A. Greenberg The purpose of this study was to determine whether any changes would occur in the biological activity of aque- ous solutions of colchicine under various storage conditions. The alkaloid was stored in the dark for varying lengths of time at two different temperatures: a) under refrigeration, O -5°C, b) and at room temperature, 21-23OC. The chemical was then tested for biological effect. The experimental materials were: pea seedlings, Eigum sativum gar; Alaska; and the alkaloid colchicine in concentrations of 400 ppm (1 X lO‘3M) for storage, and 200 ppm (S X 10'”M) for treatment. Seedlings were treated for 30 minutes, sampled at two hour intervals, prepared for microscopic examination, and scored for effect. Results appear to indicate that colchicine may be stored in an aqueous solution for #8 hours and still be biologically active as compared to a fresh solution. Colchicine stored beyond #8 hours seems not to be as bio- logically active as the fresh material or that stored up to 48 hours. CHANGES IN HIE BIOLCGICAL ACIIVIPY CF CHE ALKALCID COLCIICINE, UNDER VARIED STCRAGE CONDITIONS, AS KEASURE BY TWO CYTOLOGICAL EFFECTS ON PISUN SAPIVUN EAR. ALASKA. BY Edward A. Greenberg A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of KASEEZ OF SCIENCE Department of Botany and Plant Pathology 1962 3 J}, y! (:9 Q X I u . 1 ,3/ f (/ i. I/ . I ‘I , ACKNOWLEDGEMENTS My sincere thanks are extended to Dr. G. B. Wilson, for his patience in teaching me the principles and funda- mentals of research, and for his guidance throughout my training and research. To my colleagues in the cytology group at Michigan State University for their collaboration and aid during the course of this investigation. To Dr. J. C. Elliott for his aid and unlimited advice throughout the course of this study. Special gratitude is expressed to Dr. Jack Van't Hof, who had the patience to train me in the laboratory tech- niques necessary for the undertaking of this investigation; and for serving as a guide in my start in the field of biological research. To Michigan State University, who made it possible for me to extend my education by financial aid through a teaching assistantship. Finally to the American Cancer Society, for the partial defraying of research expenses. ii TABLE OF CC 17?"? . PAGE INTRODUCEIGN . . . . . . . . . . . . . . . . . . . . l LITERABURE REVIEW . . . . . . . . ... . . . . . . . 3 MATERIALS AND HEFKCDS . . . . . . . . . . . . . . . 10 OBSERVATIOHS . . . . . . .-. . . . . . . . . . . . . 13 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 26 SUHKARY . . . . . . . . . . . . . . . . . . . . . . 30 APPENDIX . . . . . . . . . . . . . . . . . . . . . . 31 BIBLIOGE‘IAPFTI o o o o o o o o o o o o o o o o o o o 0 1+1 iii IABL I. II. III. IV. VI. 3:“ ‘ ‘ A V IfiC k4 L w; ii}. 4—1—35) PAGE Data on the normal post-proghases, clumps, scat- ters, and the per cent effects of the fresh and stored colchicine solutions . . . . . . . . . . 32 Data on per cent effects and average ratios for each storage period and its fresh control . . . 3% Areas of the clump and scatter curves, represent- ed as 20 squares to the inch . . . . . . . . . 35 hitotic indices for the untreated controls, fresh nd stored colchiCine solutions . . . . . . . . 36 Colchicine indices of effect for the fresh and stored colchicine solutions . . . . . . . . . . 38 Clump and scatter data for the two sequences of experiments, based on 200 post-prophases per thousand cells scored at random . . . . . . . . 39 LISP CF TEXT FIGURES FIGURE PAGE 1. PrOposed structural formulae for Colchicine . h 2. Per cent effects for the fresh and 12 hour stored colchicine solutions . . . . . . . . . 15 3. .Per cent effects for the fresh and 48 hour stored colchicine solutions . . . . . . . . . 16 H. Per cent effects for the fresh and 504 hour Stored colchicine solutions . . . . . . . . . 17 5. Average ratios for the fresh and stored col- chicine solutions . . . . . . . . . . . . . . 18 6-11. Clunp and scatter curves for the first sequence of experiments . . . . . . . . . . . 20 12-13. Clunp and scatter curves for the second sequence of exneriments . . . . . . . . . . . 22 1%. Indices of effect for the second sequence of experiments . . . . . . . . . . . . . . . 2% la. Clunp and scatter curves for the fresh and stored solutions of the second sequence of experiments . . . . . . . . . . . . . . . HO NERODUCTION This study is an attempt to ascertain whether physico- chemical changes which may occur in colchicine solutions over a period of time, are measurable by visible changes occurring in a living system when exposed to the alkaloid. The number and kind of chromosomal aberrations (clumps and scatters) present in the meristematic tissue of gisgm after treatment with colchicine serve to indicate the-biological activity that remains following any changes of physico- chemical nature. The first step was to store the compound under various conditions which would normally be employed in any research laboratory-for seemingly unstable compounds; storage of the compound in powdered form or solution, a) in the dark at room temperature, b) in the dark under refrigeration. The first question asked would be, "Does-the compound remain stable either in the dry powder form or in solution when stored under 'normal' laboratory conditions for varying lengths of time?" Next, if any variations were detected, "How long may the compound be stored before any detectable changes are either eVident or of major signifi- cance?" It has been reported on several occasions that the compound is unstable, especially in aqueous solutions, as evidenced by changes in biological activity, but specific data supporting the observations are apparently lacking. It is important to know the answers to the questions 1 posed. In order to insure valid results, one must know with some assurance that the tool being used has some semblance of reliability. The results of the study indicate that the alkaloid is not as effective after a time period of storage, under the experimental conditions tested. The study suggests that more critical biochemical studies are needed, especially in obtaining a better method of measuring small quantitative changes in the relative active concentration of the com- pound. Further, studies on the effects of light should be performed and these correlated with spectrOphotometric observations. LITERATURE REVIEN Colchicine is an alkaloid which is found in several members of the Liliaceae, especially in the species Colchicum autumnale, from which the compound is most commonly extracted. This compound has been used as a drug since ancient times, being mentioned in the writings of the Egyptians. The drug was not used in a pure form, but was administered either in the form of corn fragments, entire seeds, or in a powdered form of these. Eigsti and Dustin (1955) assume that historically colchicine was used in treating gout, rheumatism, and several other ailments. They also state that the com- pound probably derives its name from the land of Colchis at the eastern tip of the Black Sea, in the Balkans. Complete historical reviews of the compound may be found in Eigsti and Dustin (1955), and Hyypio (1957). The chemistry of colchicine has not been entirely analyzed. A good description of the chemistry of the alkaloid appears in a section of Eigsti's and Dustin's book "Colchicine", contributed by London. Other investigators mentioned in the literature are: Cernoch, Malinsky, Telupilora and Santavy (1954), Rapaport and Williams (1951), Lettre (1949), and WOOd (1957). I One of the known facts about colchicine is that it is one of the trOpolones, being a tricyclic alkaloid. The first isolation of the alkaloid was made by Houde (1587). Windaus (1911-1924) analyzed the compound and suggested a formula for it which contained a partially reduced phenanthrene 3 ring. Dewar (1945), suggested that one of the end rings was a tropolone methyl ester. This has now been incorporated into the structure, and is considered correct (Fieser and Fieser 1956). ' Rings B and C of the tricyclic colchicine molecule are believed to be seven-membered; both are readily rearrangeable to six membered aromatic rings. Colchicine may also be converted to one of its isomeric forms through a tautomeric transformation. This latter derivative, isocolchicine, has been found by Steineger and Levan (19H7), to be 100 times less active than colchicine. Fig. l-Proposed Structural formulae for Colchicine: A. Windaus (1924‘ B. Dewar (1946), C. Isocolchicine, Dewar (19‘+5). D. herck Index (1960). s. huldoon (1950). 5 Colchicine is sensitive to ultraviolet light, being isomerized in aqueous solutions. The isomeric forms are the so-called: alpha, beta and gamma lumi-colchicine. (Loudon, 1955). Temperature has also been fOUnd to affect the c-mitotic* (colchicine like mitosis), activity of the alkaloid. Eigsti and Dustin (1955), mention that temperature effects are not well understood, but that at higher temperatures toxicity and resultant cell destruction occur in warm blooded animals. Colchicine, then, appears not to be very stable in aqueous solutions exposed to light and high temperatures. Solutions have been reported to lose 20 per cent of their activity after five weeks. (Eigsti and Dustin, 1955). Powell (1951), mentions that the alkaloid maintains its activity rather well, when refrigerated. Van't Hof (1960), suggests that colchicine solutions should be prepared five minutes prior to being used, because the compound deteriorates rather rapidly. Wood (1957) states that colchicine is rather stable in pharmaceuticals. His work provides no evidence to support the view that mixtures containing colchicine may lose appreciable amounts of pharmacological activity on storage for two months. Many studies have been made on the effects of col- chicine on mitosis in single cells, multinucleated organisms, no *c-mitotic: The ability to induce disturbances and/or inhibition of spindle activity with subsequent production of a) scatters, associated with partial effect; b) clumps, associated with full reaction; with recoverable multinucleate and polyploid cells, (Hadder and Wilson, 1958). 6 cleaving eggs, and synchronized pOpulations. (Gaulden and Carlson, 1949-1950; Scherbaum, 1960; Van't Hof, 1960;...) Advantage has been taken of the ability of colchicineto cause polyploidy (Gavaudan, 1937; according to Eigsti and Dustin 1955), this effect of the compound On living tissues became an important practical application in Agriculture with the work of Blakeslee and Avery (1937) and Nebel (1937). Van't Hof, Wilson and Colon (1960), took advantage of this phenomenon to tag a synchronous pOpulation of dividing cells, thus rendering them distinguishable from the other cells in the meristematic tissue by having a double amount of chromatic material. They were then able to study the effect of certain chemicals upon cell division. Cell division may be divided into two major periods the interphase stage characterized by a high metabolic activity, and the stage of active mitosis characterized by morphological and positional changes of the cell's nuclear components. Mitosis (Karyokinesis, Swanson 1957, according to D'Amato (1950), may be subdivided into three essential stages: the duplication of genetic material in the nucleus of the cell, the formation of the mitotic spindle and the movement and equal distribution of the genetic material to the poles of the cell, and finally the separation of the cytoplasm by the formation of a membrane between the two daughter cells. Wilson and Morrison (1958), state that mitosis depends on three things: 1) a series of changes in the prOperties 7 of the nuclear membrane; 2) the development and organization of the spindle material; 3) finally, the Cleavage of the kinetochore. They go on to say that any substance that is employed to induce changes in the mitotic process should be of a concentration that is within the physiological range and that the effect be reversible at some detectable level. Such a substance may be referred to as an antimitotic (Wilson and Morrison, 1958), or mitotic poison. D'Amato, 1950 says, "that a knowledge of the stages of mitosis is important for an understanding of the action of these poisons". The first observations on the effects of colchicine from a cytological point of view were made by Pernice (1889). He noticed an abundance of cells in division and several abnormalities not commonly occuring in normally-dividing cells. He also noted that the compound had a blocking effect on mitosis. Several other investigators (Dixon 1905, Dustin 1934, Lits 1934) were of the belief that colchicine in some manner stimulated cells into mitosis. Ludford (1936), was of the Opinion that the apparent increase of mitotic divisions was not due to a stimulation, but to an accumulation of mitotic figures. This, he thought, was a result of the failure of spindle formation. It has been found that colchicine is either effective in destroying the spindle or in interfering with its develop- ment (Gaulden and Carlson, on grasshoaper embryos, 1949-1951, and Levan, 1938-1954, and Hindmarsh, 1952 on Alliun root tip cells). 8 Gaulden and Carlson'(1949-1951), Brachet (1957), and Wilson and.Morrison (1958), have reported that the action of colchicine on the formed Spindle is to change it from a fibrillar to a corpuscular structure. What is not known is the specific manner in which this is accomplished. Levan (1939-19#0), Eigsti and Dustin (1955), and D'Amato (1959), are of the opinion that the amount of spindle destruction is dependent upon the concentration of colchicine. Gaulden and Carlson (1951) and Eigsti and Dustin (1955), state that there is a direct relationship between the parti- cular form a spindle will take and the characteristic meta- phasic pattern that will develop after treatment. Examples of the metaphasic patterns that will develop are: stars (Pernice, 1889), exploded metaphases (Levan, 1938), multiple stars (Levan, 1938), clumped metaphases (Barber and Callan, 1943; Berger and Witkus 1943). Hadder and Wilson (1958), report that aberrations observed (clumps and/or scatters), are dose and time dependent. The primary action of colchicine, then, is the inacti- vation of the spindle, which in turn causes many secondary effects, such as those already mentioned. In medicine, colchicine has been used with some suc- cess in the treatment of gout and some types of cancers; the objectionable qualities of the compound have been that it is toxic and persists in the human body long after adminis- tration. (Eigsti and Dustin, 1955). This seems to indicate 9 that it is either slowly metabolized, combining in vivo with substrates to form toxic compounds or that it is slowly eliminated from the body. Trezzi and Balin (1956), have found, working with Pisum sativum apical shoots, that colchicine interacts with some of the proteins; and as the pH is increased the degree of absorption also increases. Absorption of the alkaloid was found to decrease by using ascorbic acid. Wilson and Morrison (1958), found that ascorbic acid inhibited the colchicine reaction, and that this was pH and concentration dependent. MATERIALS AND METHODS GENERAL EXPERIYENTAL PROCEDURE The eXperimental material used in this investigation was the meristematic region of the root tip of the garden pea, Eiggm gativum var. Alaska. The peas were supplied by the Ferry Morse Seed Company who guaranteed that the seeds were free from chemical treatments of any sort. The colchicine used was obtained from theCLight's Chemical Company Ltd., Colnbrook, England. Two experimental sequences were performed. First colchicine solutions were stored for 12, 24, 48, 96, and 504 hours (3 wks.) at 5°C; under relative dark conditions. The second sequence consited of comparing the effects of a colchicine solution stored in the dark at room temperature (21-2300) with those produced by a solution under refrigeration (O-5OC). Each aged run in the two sequences of experiments was compared to fresh treated and untreated controls, by recording the number of clumps and scatters for each storage period and comparing them to the numbers obtained in fresh controls. The clumps and scatters were either recorded per one thousand cells scored at random or per two-hundred post- prophases. Glassware was sterilized in an autoclave, and colchicine solutions were filtered in a Seitz-filter for the second ' sequence of experiments, after it was found that solutions stored at room temperature supported a fungal organism. The powdered form of the alkaloid was divided into 10 11 equal portions, carefully sealed in vials, placed in a dessicator and stored in the dark under refrigeration. Each portion of colchicine was mixed with an appropriate amount of distilled water, prior to an experimental run, to give a 400 ppm (1 X 10'3M) stock solution. A portion of the latter was used to treat the fresh controls in each experi- ment, after being diluted to a 200 ppm (5 X lo‘uh) solution. The remaining stock solution was stored for a predetermined length of time, then used to treat pea seedlings after being diluted to a 200 ppm solution. Untreated controls were run in each experiment in order to provide a measure of any variations that could have occurred due to treatment. Peas were allowed to soak in distilled water at 25°C in a germinator prior to germination. They were then rolled in moist paper toweling and placed upright in beakers con- taining approximately an inch of distilled water. To pre- vent excessive evaporation from the toweling, wax paper was placed around the rolls. The peas were then placed in the germinator at 25°C for 36 hours; at the end of which time the primary roots of the seedlings were of an adequate length (2.5-3.0 cm.), to be used in the eXperiments. Seedlings were suspended on A inch waxed wire equili- brated meshes and (allowed to stand in a nutrient solution) for two hours. After treatment they were maintained at 22.5°C, in a water bath. Solutions used during equilibration, reatment and as a nutrient were filter-aerated at all times. 12 After equilibration the seedlings were placed in a mixture of nutrient and 200 ppm colchicine solution for 30 minutes, then washed and transferred back to beakers containing only nutrient. Samples were taken two hours after the initiation of treatment; additional ones being taken every two hours thereafter, for ten hours. At each sampling period five primary roots were excised from the seedlings and placed in vials with fixative. Pienar's fixative, which consists of a 6:3:2 mixture of absolute methanol, chloroform, and prOpionic acid, was used throughout the investigation. Each vial was coded in the same manner as the beaker from which the seed- lings were removed. The vials were then evacuated for ten minutes, capped and stored in a refrigerator for at least 24 hours. The root meristems were prepared for analysis by the Feulgen "squash" technique after hydrolysis in IN HCL at 60°C for 18 minutes. The slide preparations were dehydrated in 9:1 tertiary-butyl alchohol and absolute ethyl alcohol for at least 12 hours, then made permanent with.diaphane. The slides were then examined and scored for effect. OBSERVATIONS Colchicine generally produces two abberant configurations in the meristematic tissues of Eigum: the scattered metaphase (c-metaphase of Levan, 1938), produced by treatments with low doses, and short treatments with high doses. The second abberant configuration is the clumped metaphase (Barber and Callan, 1943), produced by extended treatments with low doses, and with high doses. (Hadder and Wilson, 1958). Thus, Hadder concluded that "scatters and clumps express different degrees of the same primary effect, and that the degree of effect is dependent on dosage; which may be expressed as duration of exposure to a given concentration." Van't Hof (1960-1961), determined what concentration and duration of treatment produced the best reaction and recovery from the effects of the alkaloid. According to ,him the reaction time is that period during which chromosome configurations (clumps and/or scatters) leading to tetraploidy are observed in significant numbers, and recovery period the time interval from the peak reaction to that point where 10 or less per thousand figures showing effect are observed. He concludes, "ideally, the duration of treatment should be as short as is consistent with both a rapid recovery from colchicine effect." Thus, any changes in the compound's effectiveness should be easily detected, by using a constant eXposure concentration (5 X lO'HM) and time (30 min.). Since the reaction period is usually quite constant and does nbt show change (Van't Hof, l3 14 1960—1961), the best measuring device whould seem to be the recovery period eXpressed as per cent effect (Number of clumps and scatters/total post-prOphases observed). Comparing data for the storage periods of 12, 48, and 504 hours (3 wks.), and representing these in graphic form (Figures 2,3,and 4), it may be seen that in aging from 12 to 48 hours the recovery curves are more or less identical. However, the solution stored beyond 48 hours appears to show considerable change, especially from 4-6 and 6-8 hours after the initiation of treatment. An observation of considerable interest is that re- vealed by data for Figure 2. It seems that the solution stored 12 hours is more effective than its fresh control; this was also seen, but to a lesser extent, in the solution stored 24 hours (Table I, appendix). Figure 5 was constructed to show changes when the average ratios of each storage period are compared. These average ratios were obtained by dividing the average per cent effect for all the points of the stored solution by the average per cent effect for all points of its fresh control. This graphic form shows that from O to 48 hours there has been no apparent change, however, from 48 hours to three weeks a drop of .51 units is observed. A more sensitive measure of change was obtained by plotting the number of clumps and scatters per thousand cells counted at random. Each point on the graph represents the average number of four slides per sampling period. % EFFECT 90‘ 80* 70* 60’ 50* 30~ ZCN IO? 15 Figure 2. 4 s TINE IN HOURS % EFFECT 90- 8C? 70 60' 50* 40- 30' 20 IO‘ 16 4 6 8' IO TIME IN HOURS # L NL Figure 3. 17 90- 8C% 70- 60- L O a. Homhmm R d 4. 30* 20' IO' é 8 IO TIME IN HOURS 4' Figure 4. 18 W¥§-m. 0- -—-_®._-_. mzHa moamoam ,“mmtém . «IE. d H ((m u o-@—-—o “.mmfiwm -—-—-—-—-—o-—-—-—- .mmzwi ..-—.-—-——-—-——--—@-_.—. .m madmfim EAV HS'HHfl/GSHOIS OLIVE EOVH 19 The curves are shown in Figures 6-11 (Table I, appendix) and represent points for both the fresh and aged eXperimentals. Areas under the curves were obtained by countingthe squares, 20/inch, (Table 111). At three weeks the compound shows greater change in its ability to produce the cytological effects at the concentrations used. Observations based on the clumps alone produced curves that are flatter and com- pressed. Some measure of the effects of storage upon the dry powder form of the chemical were also obtained. Data collected over a three month period from the portions of the dry pow- der indicate, in comparing Figures 8 and 6 to 10, that the fresh controls may diminish in activity over a three month period. The second sequence of experiments involved a com- parative investigation, between the changes in biological activity of a solution stored‘at room temperature and one placed under refrigeration. Both solutions were stored in the dark for three weeks. The powder form of the chemical was obtained as a fresh batch from the Light's Company. The eXperimental procedures followed were the same as those in the first sequence. If, in this case, one again compares the clump and scatter data, it may be seen that there has been some change in the active concentration of the com- pound after three weeks. (Figures 12 and 13). _ Van't Hof (1960), reported that different batches of the chemical are never the same in activity. 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Data on Per Cent Effect and Average Ratios for each Storage Period and its Fresh Control. 0 agg12 Hour Stored Colchicine. Hrs. p effect fresh effect stored ave. ratio 2 83.10 89.31 1.07 4 58.88 61.14 1.04 6 48.48 55.69 1.15 8 15.61 40.71 2.61 10 7.86 8.55 1.02 Ave. 1.39 0 and 24 Houg Stoged Colchicin . l‘iI‘S. 2 89.61 87.7 .97 4 6 .38 62.6 .96 6 3 .23 49.44 1.29 8 41.57 22.81 .55 10 11.33 7.96 2.01 Ave. 1.1 0 and 48 Hour Stored Colchigine. iirs. 2 93.41 79.11 .85 4 71.17 53.94 ..76 6 51.62 56.81 1.10 8 14.28 23.36 1.99 10 5.92 15.69 2.22 Ave. 1.39 0 and 96 Hour Stored Colchicine. Hrs. 2 91.67 90.00 .98 4 73-33 72-92 ~99 6 63.75 64.94 .94 8 8.40 42.:2 .82 10 2 . 7 1 . 7 .gg Ave. 0 i e Hrs. 2 91.54 85.63 .93 4 69.34 57.14 .82 6 46.87 23.87 .51 8 22.05 31.18 1.41 10 25-53 18~13 Ave. 34 Table III. Areas of the Clump and Scatter Curves, represented as 20 Squares to the Inch. Brash Clumps Scatters Easel! Clumps Scatters .Etseh Clumps Scatters £232.82 Clumps Scatters 803 324 599 515 1.24 397 460 545 Stgrgd 12 Hogzg Clumps Scatters Stored 48 Hours Clumps Scattes 785 500 382 506 fitored 3 Wkg. (1st.Seduence) Clumps Scatters 243 525 Stored 3 Wks. (Refrig. 2nd. Seq.) Clumps' Scatters Storedl3 Wks. Room Temperature (2nd. Seg.) Clumps Scatters 183 607 341 503 35 05.00 05.55 05.:0 50.:0 0. 00..0 50.0 55.55 50.05 0 50.00 00.0 55.00 50.05 0 05 .00 55 .00 00.50 55 .05 : 55.00 05.00 55.50 50.05 mm . 0 mhzom 0: 009000 Hoppcou £0095 Monacoo 0opmmaunb . wepwoapCD 05.:5 50..0 00. 50 00.00 0. 00. 00 55.:0 55. 00 00.05 0 05. 05 05.50 05. .0 50.:5 0 55. 05 50.00 05 .50 55.:5 : 55. 00 55.05 05.05 55.00 0 .mnm madom :m camOpm Homusoo £0000 Hoppcoo wepwmpucb oepmempCD 00.00 55.00 05.05 00.: 0. 50..0 05..5 00. 00 55.0 0 50.50 00.05 05. 00 00.00 0 50.55 55.00 00.55 55.0 : 50.05 50.05 55.50 55.0 mm . 0 00:02 N. 009000 Hompcoo swank Honpcou woummmpCD ompmonuCD .mcofipsaom 0000000000 000000 0:0 00005 .00000000 000000000 000 00 000.00H 0.000.: .>. 00000 /O 33 55.00 00.00 05.:0 00.05 00.0: Om.©or 55..0 00.0w OO.@5 Ow.Fm 0A QUQW cum Fv oo.mw 00.:5 55.00 00:00 00 000000 .500005000 .00N .0000000 0000 .000 ..0E00 .e00 .003 0 000000 .003 0 000000 05.3 55.00. 00.00 05.55 50.50 55.05 50.55 00..0 00.00 55.00 mN.©oe 00.05 05.00 00.:0 50.00 0000000 00000000: 000p£oo 00000000: 0000000 00000000: .003 0 000000 55..0 55.50 55.00. 00.:5 05.55 £0000 50.05 00..0 00.00 00.55 05.05 £0000 50.55 00.00 50..0 55.00 05.05 £0000 00.05 00.00 50..0 05.00 55.05 00.00 mN.FoF Om 3:5 55..0 0N.om 05.:0 50.00 05.00 55.00 50..0 .A.0.pcoov .902 0000:00 00000000: or w o +— N .m0m 0000000 000000002 or w w J N .00: 0000000 00000000: or w .l +~ N 00: 0000000 00000000: .>H 0H£0H Table V. Fresh Hrs. 2 1+ 6 Fresh Hrs. Fresh Hrs. 37 Colchicine Indices of Effect for the Fresh and Stored Solutions. Stored 5Q Hours 1.78 3 1.70 1.27 1.36 068 .93 £322.90; 3 Eka- (W) 1.70 1.37 1.07 .80 .H3 .26 £19200 3 M02 (.___2nd- __Q_Se 0301 322022322. 1.7% 1.30 1.01 .68 .50 3 .hh fifigged 3 Egg; (Refrigerated, gag. §gg.) 2 1.35 H 1.24 00.50 00.00 55.05 55.00 55.00 05.00 .A0000000m .00N .000000000000mv 50.00 00.00 50.00 05.:— 00.00 55.00 55.05 50.00 00.00 00.00. 50.00 50.000 my .A0000000m .00m ..0009 Soomv .mxz m 000000 00.0: 05.0 05.M5 55.00 05.05 50.55 50. 5 00.00 00.00 05.500 05.5: 00.550 .A0o00000m 000000 .003 m 000000 55.000 00.00 00.000 00.00 05.05 05.500 00.50 55.00 00.05 . 50.050 50.50. 50.500 00000000 0000H0 00000000 000000 000om 0: 000000 .000000 00 000000 00000 00000009 000 00000000mu0mom 00m 00 00000 0000000000xm 00 000000000 039 000 000 0000 0000000 000 00000 0 J N .00m .003 0 000000 0 IT— 0 .000 00000 .00m 0000b 0 +~ N .000 00000 .H> 0HD0H 39 COMPARATIVE EXPERIMENT (COLCHICINE STORED 3 WKS.) IBO [60 \ I: O R) 0 CD 0 CLUMPS AND SCATTERS/ZOO POST-PROPHASES cs ES 0) C) 20’ r 1 _ Figure 1a. 0—0 CLU] :Ps FRESH SOLUI'ION ®---0 SCAI‘I’SRS " CLUI IPS REFRIG“RAI’ED SOLULION --- SCAL‘EERS " 0—0 CLUMPS ROOM-TEMP. SOLUTION 0----o SCATTERS " " 4 4 TIME (hrs) 0‘- BIBLIOGRAPHY Brachet, J. 1957. Biochemical Cytology. Academic Press New York. 535 Pages. Berger, C. A. and C. R. Witkus. 1993. A Cytological Study of C-Mitosis in the PolysOmatic Plant Spinacia gleracea with Comparative Studies on Allium ceoa. Bull. Torrey Bot. 29: 457-s66. D'Amato, F. 1959. C-Mitosis and Experimental Polyploidy in Plants. Genetica Agraria fig; Eigsti, O. J. and P. Dustin Jr. 1955. Colchicine in Agriculture, Medicine, Biology, and Chemistry. Iowa State Press. #70 pages. Elliot, F. C. 1958. Plant Breeding and Cytogenetics. McGraw—Hill Book Co. Inc. 395 pages. Fieser L. F. and M. Fieser. 1956. Crganic Chemistry. D. C. Heath and Company, Boston 3rd. ed. 640-643. Gaulden, K. E. and J. G. Carlson. 1949. 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Colchicine-Induced C-Mitosis in fwo House Ascites Tumors. fiereditas fig: 1-64. Kazia, D. 1961. Biochemistry of the Dividing Cell. Annual Rev. of Biochem. gg: 669-687.. Morrison, J. H. and G. B. Wilson. 1956. Influence of L- Ascorbic Acid on the Colchicine Reaction. Science 122: 1359—1390. Muhling, G. H., J. Van't Hof and G. D. Wilson. 1960. Cytological dffect of Herbicidal Substitutec Phenols. Weeds Q: 173-181. Nebel, B. and K. Ruttle. 1938. 1'he Cytological and G g2: 3‘90 (D netifial Significance of Colchicine. J. Hered. Powell, S. S. 1951. Comparative Effects of Colchicine and Various Nucleic hcid Salts upon Somatic Kitosis. (M.S. thesis, Kichigan State University). AZ Swanson, C. P. 1931. Cytology and Cytogenetics. Prentice- Uall Inc. New.York. 596 pages. Schrader, F. 1953. The Hovement of Chromosomes in Cell Division. Columbia University Press. 107 pages. Trezzi, F. and L. Balin. 1956. SpectrOphotometric Research on the Action of Colchicine on Protein Extracts in-Vitro. Atti. accad. nazl. Lincei. Rend., classe sci. fis., mat. e na . 2Q: 60-71. (Chem. Abs. 59: lOi39). Van Dreal, P. A. 1961. Kitotic Activity and Respiration -in the ii"KeiSed Pea Root heristem. (Ph.D. thesis, Kichigan State University). Van't Hof, J. 1951. Rate Changes of the Yitotic Cycle. (Ph.D. thesis, Michigan State University). Van't Eof. J., G. B. Wilson and A. Colon. 1960. Studies on the Control of Mitotic ncitivity. J-'he Use of a Synchronous Population of Cells in the Yeristem of Pisum sativum. Chromosome 11: 313-321. Wilson, G. B. 1960. fhe Study of Drug Effects at th Cytological Level. Intern. Rev. of Cytol. 9: 293- 304. Wilson, G. B. 1959. Fourth Huskins heuorial Lecture. ian J. Genet. and Cytol. 1: 1-9. Studies on the Control of Kitotic hctivity. Cane Wilson, G. B. and C. C. Bowen. 1951. Cytological Effects of Some Kore Antibiotics. J. Hered. egg: 251-255. Lt3 Wilson, G. B. and J. H. Horrison. 1958. Mitotic Activity and Behavior as an Index of Chemical Effect. The Nucleus 1: 45-56. Wilson, G. 3., J. H. horrison and N. Cnobloch. 1959. Studies 'on the Control of Mitotic Activity in Excised Roots. J. of Biophys. and Biochem. Cytol. 5: 411-420. Wilson, G. 3., P. fsou and P. Hyypio. 1952. Variations in Vitosis. J. Hered. 33: 211-215. Wood, D. 1. 1957. Stability of Colchicine in Pharmaceuticals. Parm. J. 128: 188. 0.9.51. 7 .ffiv . '3 .9 w-M