THE wlITOTIC EFFECT OF TECHNICAL LINDa NE ( r-HEXACHLOBOCYCLOHHXAi'JE) By Te May Tsou Ching AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology Year 1954 s'’ *? -— Approved / ProQuest Number: 10008280 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008280 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Ab£TfviiCT Tiie main object of tnis investigation was to study the abnormal cytological behavior inauced by the insecticide, technical Lindane, which contains 25% wettable mixture of Y—hexachlorocyclohexane and an inert carrier; in order to explain some aspects of the mechanism of mitosis and some hazards encountered in practical application. The root tips of onion bulbs and pea seedlings were treated in 0.1% suspension of the insecticide. The mitotic indices of these treated meristemic tissues remained essentially the same as "zero hour controls1’. The prophase frequencies gradually dropped, metaphase frequencies signifi­ cantly increased, whereas post-iaetaphase frequencies remained unchanged in continuous treatments. This investigation further suggested that there are at least two more or less independent components involved in spindle function: 1. a dipolar cytoplasmic orientation possibly characteristic of the cell at any stage, and 2. a nuclear component inherent in and directed by the chromosome or the kinetochore thereof. The insecticide is capable of inhibiting both components, thus inducing so-called ”c-mitosis”. The immediate effects of the insecticide upon mitotic stages are con­ traction of chromosome, possibly breakdown of nuclear membrane, partial inhibition of ’’spindle function” and failure of cytokinesis * As treatment is prolonged, over contraction of chromosomes and complete destruction of the spindle mechanism occurs. 2 Inositol antagonism and radiomimetic effects of this insecticide have not been demonstrated in this investigation, thus further research is desirable* While Lindane is theoretically a good polyploidizing agent, there are a number of practical difficulties which reduce its potentiality below that of colchicine. The final proaucts of atypical mitosis induced by the insecticide s.re polyploid cells and aneuploid microcytes. The former are di^nt cells, but possess a much slower growth rate than that of normal ones* latter usually tend to be lest, viable. The Therefore the treated meristemic tissues are much reduced as far as number of cells and rate of growth are concerned. The insecticide also causes "c-tumour" in the enlargement zone of treated root-tips. Most workers believe that this effect is independent of "c-mitosis" and probably is due to disturbance of hormone polarity at the cellular level. This disturbance causes abnormal growth, such as swelled shoot and root apex, twisted parts, lack of root hairt, distorted leaves*etc* The precise, correlated normal physiological balance of some sensitive plants is definitely affected, when they are grown in soil con­ taining the insecticide either as soil treatment or residue of spary. BlbLiOGhaBHI Boswell, V. R. 1912. Residues. Soils* ana Plants. Book of U.S.D.A.:284-297. Insects. 1952 Year D fAmato, F. 1949. Sull*impiego Bel Garmiiess.no Coine Anente Poliploidizzante. (Use of gammex&ne as a polyploidizing agent) Caryologia 1 (2):209-222. Kostoff, D. 1949. Atypical Growth. Abnormal Mitosis. Polyploid?/ and Chromosomal Fragmentation Induced by Hexacillorocyciohexane. Nature 162:845-846. Nybom, N. and B. Knutsson, 1947. Cepa. Hereditas 33:220-243. Investigation on c-mitosis in Allium Ostergren, G. and A. Levan, 1943. The Connection Between c-mitotic Activity and Water Solubility in Some Monocyclic Compounds. Hereditas 29:381-443. Scholes, M. E. 1953. The Effect of Hexachlorocyclohexane on Mitosis in Roots of Onion and Strawberry (Fragarja Vesca). Jour. Hort. Sci. 28(1):49-68. Wilson, G. B., M. E. Hawthorne, and T. M. Tsou, 1951. Spontaneous and Induced Variations in Mitosis. Jour. Hered. 42:183-189. , T* M. Tsou and P. Hyypio, 1952. Variations in Mitosis. II. The Interrelation of Some Basic Deviations. Jour. Hered. 43:211-215. THE MITOTIC EFFECT OF TECHNICAL LINDANE ( T-HEXACHLOROCYCLOHJiXANE) Te May Tsou Ching A DISSERTATION Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of . DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1954 , '/ Te May Tsou Ching Candidate for the Degree of Doctor of Philosophy Final Examinations: May 18, 1954, 10-12 a .M. Botany Seminar Room Dissertation: The Mitotic Effect of Technical Lindane ( Y-hexachiorocyclohexane) Outline of Studies: Major Subject: Botany (Cytology) Minor Subject: Zoology Biographical Items: Bora: January 9, 1925, Soochow, China, Undergraduate Studies: Graduate Studies: Experience: National Central University China, 1940-44 Michigan State College, 1949-54 Teaching Assistant - National Central University,1944-47 Graduate Assistant - Michigan State College, 1951-54 Member of; Genetic Society of America Sem. Bot. Society of the Sigma Xi Xi Sigma Pi Fraternity i 365824 ACKNOWLEDGMENTS The writer wishes to give her sincere gratitude to her colleagues in the cytology group at Michigan State College. Their encourage­ ment and discussion have made this investigation gay and interesting. To the leader of the group, Dr. G. £. Wilson, the writer is espe­ cially indebted for his advice pertaining to the study and for his assistance in the preparation of the manuscript. To Mr. Philip Coleman, thanks are extended for his invaluable photographic work. Appreciation is extended to the All College Research Committee for defraying many of the research expenses, as well as to the Agricultural Experimental Station of Michigan State College for a Graduate Research Assistantship which the writer held during her final year of graduate virork. ii TABLE OF CuNTENTS Page INTRODUCTION........................................... 1 MATERIALS AND METHODS..................................5 I. II. III. The Insecticide........... .. .............. 5 Experimental Procedure..................... 6 Cytological Examination ...................... 8 OBSERVATIONS.......................................... 10 I. II. III* IV. Variation in Mitotic EffectsWith Duration. . . . 10 Aberrations...................... .......... 27 Comparison of Results..................... 33 The Abnormal Mitosis Inauced byTechnicalLindane 36 DISCUSSION............................................ 41 I. Mitotic Aspect............ II. Chromosomal Fragmentation................. 44 III. Polyploiaizing Effect.....................45 IV. V. VI. Inositol Antagonism . . 41 ..................... 46 Difference Between Lindane andColchicine. . . . 47 Explanation of Phyto-responses............. 50 SUMMARY.............................................. 56 BIBLIOGRAPHY.......................................... 58 PLATES.......................................................63 APPENDIX..............................................88 1 INTRODUCTION Numerous reports have been, written concerning the phytotoxic Lty of the insecticide^technical Linciane or technical benzene hexachloride ,since the insecticidal value of the chemical was verified by blade (19.45)* Sensitivity to the insecticide differs in different species, even the same varieties of plants react differently at different ages, locations or by different handling methods (Boswell, 1952; Ashby, 1950). Thus the phytotoxicity of any insecticide is a representation of the balanced effect of all factors involved in field work; such as genetic potentiality of the plant in ques­ tion; the soil; the climate; and the method of treatment,etc..On the whole, the phytotoxicity of this insecticide may be summarized as follows (Bos­ well, 1952; Cullinan, 1949; Ashby, 1950; Greenwood, 1949; Stitt and Evanson, 1949; and Stoker, 1948): 1. Seeds sown in soil, which contains the insecticide either as soil treatment or as residue from previous spray were injured by the in­ secticide and showed reduced germination rate. Someti:aes germination was not affected but emergence from the soil was. 2. When the insecticide was present in the soil in appreciable amounts, plants showed a definitely reduced rate of growth, reduced total growth and decreased yield (as of seed or fruit)• 3. If a spray method was used, the young leaves were easily scorched or distorted by the insecticide. Young vegetable seedlings and orchard trees were often injured by spray of high concentration. 4. The plant grown, in soil containing the insecticide usually had a poorly developed root system; tne roots were numerous, short, stubby, thickened, and virtually witnout root hairs. Thus root vegetable crops were definitely damaged by technical Lindane. 5. The edible crops were often off-flavored by the presence of technical Linatuie in soil or as a spray. The mild flavored vegetables such as peas, beans, lettuce and potatoes especially were tainted, but altered taste was not obvious in strong flavored onion or rauishes. If technical Lindane is used in a soil treatment or spray iur above­ ground parts, it eventually accumulates in the soil. Since it is insol­ uble in water, it remains ^uite stable in the soil and little or no de­ composition occurs. Owing to the volatility of the chemical present in the insecticide and to its absorbtlon by organisms in soil, the insect­ icide decreases in amount. Some workers reported that every year the accuraitlated amount decreased about 10 percent, if no additional applica­ tion was made (West and Campbell, 1950). The pure chemical has been used for theoretical study by Ostergran and Levan (1945). They stated that it caused colchicine mitosis or c-mitosis, and that the c-mitotic property is related to water solubility and tne relationship is an inverse one. In addition, there is a positive correlation between c-mitotic activity and lipid solubility. Nybom and Knutsson(1947) confirmed the above statement ana further proved that there is a negative correlation between the melting point of various isomers and their c-mitotic activity. They also started investi­ gation of the insecticide "666" which causes essentially the same mitotic 5 effects as benzene hexachloride. Kostoff (1948 and 1949) reported that atypical growth, abnormal mi­ tosis, polyploidy and. chromosomal fragmentation were induced by hexachlorocyelohexsne. He applied commercial powaer to seedlings of twelve species of angiosperms; Zea mays, Trlticum vulharer X* monococcum, T. compacturn, Secale cereale. betaria italica, Panicum miliaceum. Heiianthus annus. Cropis capillarIst Vicia faba> X* sativa and Brasslea nigra. Suppressed growth and thickened shoot ana root apexes as well as cytological aberrations re­ sulted, Furthermore, he pointed out that the chemical is a cheap poly- ploidizing agent. Keo and Kundu (1949) came to the same conclusion after they treated root tips of Corchorus capsularis with gammexane. More intensive vi/ork has been done by D'Amato (1949). By immersing root tips or shoot tips of seedlings of twelve species of angiosperms in dilute ethanol solution of pure gammexane, polyploid ceils were obtained. However, germination of seeds in supersaturated solution did not induce polyploidy in either root or stem tips. practical application of the insecticide as well as a cytological study of the effects were carried out by bcholes (1985) on onion seedlings with tne insecticide. She stated that tne frequency of nuclear division was unaffected, but low doses sometimes led to exclusion of nuclear frag­ ments in subsequent cell aivision while higher aosos lea to polyploidy. Strav.'berry runners rooted in soil treated with tne insecticide showed similar cytological derangements. Owing to the interesting cytological effects of the chemical and 4 also the physiological uis tar banco s caused by practical application, tho insecticiae provia.es a challenge to botanists. Cells are universally known as basic units of organism ana any deviation of eeaiular behavior may cause physiological as well as genetic&l changes. Thus this insect­ icide has been selected for research at the cytological level., in oroer to explain some encountered hazards ana if it is poseiDle, to throw some light on one of the basic biological problems, i.e., the mechanism of mitosis* 5 MATERIALS-ArtD m E T H O D d I. The Insecticide The molecular formula of hexacnlorocyclohexane or benzene hexachloride is C6H6Cl6> theoretically it may have eighteen isomers* but only five have been syntnesized and the names alpha, beta, gamma, delta and epsilon have been given according to their sequence of discovery* The insecticidal property of the chemical was discovered in France in 1941. In the follow­ ing year, the Labouratory of Imperial Chemical Industries in England veri­ fied the fact. Slade (1945) indicated that the gamma isomer is most effective as far as the insecteradic&ting property is concerned. Since then the compound has been used in Europe and America in various forms and under different commercial names, such as Gammexane, 666, Agrocide 7, Agtocide 3, Hexadow, Hexachlorane, "lindane” etc.. The structured, formula of Y-hexachlorocyclohexane is H The pure chemical is a volatile crystal with a melting point of 112.5° C, insoluble in water and soluble in organic solvents especially in acetone and ethanol. In a weak alkaline solution^it splits off three * moles-of hydrogen chloride to form a mixture of trichlorobenzenes. 6 (Shepard, 1951). The insecticidal property of the gamma isomer of this compound may be due to interference with the inositol CC6h 6(0H)6) metab­ olism of insects as suggested by Kirkwood and Phillips (1946). Since inositol is. a member of the vitamin B complex and also is a common metab­ olite in animals, and in addition, suppressed the inhibitory effect of y-hexachlorocyclohexane on growth of yeast and fungi, the suggestion is not an unreasonable one. The name Lindane is applied to those grades of tf-hexachlorocyclohexane which have a purity of 99 percent or better. used The insecticide.,"Lindane” in this investigation was obtained through the courtesy of the Dow Chemical Company, Midland, Michigan. It consisted of 25 percent wettable mixture of the chemical and an inert carrier and is commonly used as a general insecticide on plants and animals. Owing to its insolubility in water, the concentration of test solutions was rather difficult to a s c e r - ’ tain. Some primary experiments showed that there is no striking cytological difference between 0.01 % and 1.0 % suspensions. On the other hand the filtrate of those suspensions did not show full c-mitotic effect on meristemic cells within a reasonable time. Since only water is used for field work, a water-suspension ofQ+1% hy weight was selected for tests. I I • Experimental Procedure The root tips developed from bulbs of the common commercial yellow variety of Allium cepa L.» and from young seedlings of Pi sum sativum var. Alaska provided the meristemic tissues for the study of the mitotic effects. 7 The onion bulbs were obtained from a single crop produced by Mr. Emil Pontack of Elsie, Michigan. The pea seeds were furnished by the Ferry- Morse heed Company, and reported to be fresh stocx from a diseate-free strain of relative genetic uniformity. The onion bulbs were rooted in aerated distilled water until the ma­ jority of roots were two centimeters in length; four roots from each bulb were excised and designated as "zero hour controls". Then the rooted bulbs were transferred to treatment suspension or aerated distilled water which was used as control. The pea seedlings were germinated in moist paper towel about two and one-half' days. The roots of selected young seedlings, about two to three centimeters long,were inserted in half strength modified Hoagland's nutri­ ent solution (HusJcins and Steinitz, 1948) by means of a wax coated metal grid placed on a beaker. Constant aeration and agitation was furnished by bubbling air through capillary openings in the ends of glass tubes inserted into the solution through the grid. The air was obtained from the labora­ tory service outlets but was first rendered free of dust and oil particles by being passed through a filter consisting essentially of eighty mesh char­ coal. After six to twelve hours of nutrient solution treatment, four root tips .were collected as "zero hour controls" for each run; the rest of the seedlings were transferred into the test suspension containing quarter strength modified Hoagland nutrient and 0.1 % of "Lindane" by weight. A control run treated only with the nutrient solution was also carried out simultaneously with each test. 8 Collections were uaue at intervals up to one hour for short time treatments of onion, up to twenty-four hours for onion and pea continuous treatments. For recovery runs, twenty-four hours v.ere also used. All the experiments were carried out under laboratory conditions, with a room tem­ perature of about 24° C and an average relative hunidity of 50 percent. Excised root tips were fixed, in three to one absolute alcohol-glacial acetic acid mixture for 10 minutes at 60° C for pea, and 15 minutes for onion. Hydrolysis in one normal hydrochloric acid for nine minutes for pea, 12 minutes for onion at 60° C was followed by staining by the Feulgen technique. Squash preparations were then dehydrated over ni^ht in 95 % ethanol containing a small amount of fast green as a counterstain, and made permanent with Diaphane. III. Cytological Examination All examination of slides was done with a 90X oil immersion objective and 12.5X oculars. Critical illumination of the slide was provided by a ribbon filament lamp with a type B green filter between the lamp and the microscope mirror. The phase microscope also was used for checking the presence of nuclear membrane, spindle and cell plate. Qualitative information concerning mitotic effects was obtained by random checking right after the temporary slides were made. Careful notes were taken on apparent frequency of division figures, types of abnormali­ ties and finally evidences of poisoning effect in both dividing cells and resting nuclei. y Quantitative oata were procured by a rigia system. fetch slide was examined in horizontal stripe in order to obtain a mitotic index and a mitotic formula. The former was based on 1,000 meristernic cells, the latter was calculated upon 100 dividing cells. Data taken in this investigation are surumerized in Appendix tables 1 to 6, ana form the basis for all quantitative ana statistical analysis of the mitotic effects. 10 OBbLKVATIOi'lS I, Variation in Mitotic Effects With Duration of Treatment Any spontaneous or induced variation in normal mitotic activities of a meristernic tissue may be considered as mitotic effects of a certain disturbance- The effects may be classified mainly as changes in mitotic index, mitotic formula and mitotic behavior* The so-called mitotic index is the frequency of mitotic figures per hundred merict-emic cells. The mitotic formula is an expression which is composed of percent distribu­ tion of various stages of mitosis. And finally, the mitotic behavior is usually described by deviation from normal stages or phases which are characteristic of the normal mitotic sequence. 1. Onion: The results obtained from treatments for 15, 50, 45 and 60 minutes showed that the mitotic indices decreased then steadily increased (Fig.2); whereas the control root-tipe excised within one hour had an index similar to that of the zero nour controls (Fig. 1). As for long time continuous treatments, the mitotic indices showed a slight drop at the half-hour point followed by a steady increase up to 2 hours;there the index reached a maximum which was maintained through 7 hours treatment, after which it dropped abruptly to normal (Fig. 4). The results of the recovery after one hour treatment were rather erratic, but an ascending tendency was 11 obvious (Fig. 5). The long time continuous controls were somewhat irre­ gular; the average index, however, was close to that of zero hour controls (Fig. 3). The mitotic formula of controls remained within the range of the zero hour controls; in other words, the mitotic formula at any time.was more or less constant (Figs. 6 and 8). In continuous treatment, the prophase percentage gradually decreased and remained significantly lower than that of controls. The metaphase percentage on the other hand increased rather rapidly and remained definitely higher than that of controls (Fig*. and 9). In the materials which were recovered after treatment of one hour, the mitotic formula reached maximum deviation at one hour after the treatment, then gradually approached normal. However, the effect still remained, i. e.,the prophase percentages were lower than that of normal, metaphase percentages were higher than that of normal, and postmetaphase remained within normal range (Fig. 10). The mitotic behavior of treated onion root-tips was definitely different from that of controls. In untreated or control root-tips there were never more than 20 percent of the total of any mitotic stage (Figs. 1 and 3), but raetaphase abnomalities quickly jumped to 100 percent within one hour»s treatment and remained so provided the drug was not removed. If the drug was removed after one hour, the effect still remained but to a milder degree, and consequently some normal figures gradually appeared as "recovery" proceeded. later. Postmetaphase reacted similarly but somewhat Prophase responded quite rapidly showing' some aberration within 7 12 one hour1s treatment. However, most prophsses were iiormal shortly alter the roots were removed from treatment (Figs. 2, 4 ana 5). If one takes the mitotic inaices into consideration, mula may not be a good means for comparison. the mitotic for­ Thus it is necessary to put all data on the same basis and analyze them statistically in order to obtain a reasonably complete picture. The frequencies of prophase per 10,000 meristernic cells from 9-14 hours treatment and 1-4 nours recovery after treatment oi' one hour showed that they were statistically lower than that of normal, otherwise tnere were no airferences from those of normal* The frequencies of metaphase were signiiicantly higher than normal both in treatments and recoveries. As for post-metaphase, only 9-13 hours recovery material showed definitely, higher frequencies than normal. The divisional rates were significantly higher than controls both after £-4 hours treatments and 9-13 hours recoveries at 5 percent level (Table 1). 2. Pea: A definite drop of mitotic indices in the first six hours was obtained in continuous control pea root-tips followed by an increase at eight hours (Fig. 11). Continuous treatment of pea proauced only apparently normal fluctuation of index(Fig« 12).-The statistics showed no significant aifference in mitotic index between continuous treatments and zero hour controls. The mitotic indices however during the first four hours- of continuous control appeared to be decreasing but no difference was found after 5-8 hours (Table k). The percent distribution, oi various mitotic stages in continuous con­ trols were rather close to that oi tne zero hour controls, but further statistical analysis revealed that the frequencies of prophase in 1-4 hours were significantly lower than that of the zero hour. The frequencie of other stages were not different from the control (Fig. lb, Table k). In continuous treatments, the frequencies of prophate showed a slight uptrend at one hour then gradually decreased till 6 hours and then in­ creased again. The frequencies of metaphase, on the contrary, went down first then up steadily till 6 hours and dropped again. The frequency changes of post-metaphase were rather insignificant diagrams.ti cully and statistically. As for prophase and metaphase, the decrease and increase were statistically significant after 6-8 hours treatment* Tne micotic behavior of continuous controls was very regular, and the abnormality of any stage at any time seldom exceeded ten percent of the total percent of the stage. Prophases had the fewest aberrations and metaphases the highest (Fig. 11). In the case of continuous treatment, the prophases, comparatively speaxing, were most inert, while metaphaies changed rapidly to 100 percent aberrant within four hours., treatment* Post—metuphases also reached maximum aberrations at four hours, but more gradually. In comparison to continuous controls, even prophase showed almost double the abnormalities at any stage of treatment (Fig, 12), CUUiiWIGON OF CONikOL, UJNTINLJOUO TkEaTolENT a NLi Time (hr.) nEOuVEai tiUNb OF ONION No. oi' No. oi pro- No. oi metaslide phase per phase per 10,000 cells 10,000 cells Control 16 261± 18.6 i-4 5 270 i 17.6 5-8 4 9-14 69 ±4.7 No. of post- No. of aivimetckphs.se per aing iigures 10,000 cells /10,G00cells 116 + 10.3 446 ±28.7 218 — 44 •6** 126± 21.5 614± 55.3* 181± 49.2 232* 27.9** 157 ± 5.1 550± 78.7 4 140± 26.3 142 ± 6.2** 126± 52.8 405± 38.6 1-4 4 198 ± 10.5** 138 ± 31.7* 151 ± 24.9 488± 43.8 5-8 4 287t 30.9 134 ± 16.0** 112 ± 21.1 533± 67.6 9-15 4 250 £36.4 182 ±30.8** 1531 9.7* 585 ±_39. 7* Treatment Recovery * significant at 5% level ** significant at 1$ level lAbLE II COiviPiiRloUN OF ZERO HOUR CONTROL, CONTINNOJb CONTROL AND coNTlNUOlJO TREATMENT OF REA Tiiae No. of No. of pro- No. of meta(hr.) slici© phase per phase per 10,000 cells 10,000 cells Zero Hour Control 51 No. of ^ost- No. of divimetaphase per ding figures 10,000 cells /10,000celis 125 ± 6 . 3 131 ± 6.3 631± 25.9 371 ±57.3 154 ± 2.2 118±53.8 645 ±59. 3 299±57.1* 207 ±18.8** 104 ±21. 3 606 ±60.5 588 ±18.5 Continuous, treatment: 1-4 14 5-8 8 Continuous control; 1-4 8 353± 17.1* 110 ± 9 . 6 120±13.1 563 ±22. 3* 5-8 4 576± 69.6 124 ±16.7 120 ± 9.4 618 ± 84.9 * significant at b% level ** significant at 1% level 1C ------- Mitotic Indices as % oi control ------- % aberrations in prophase ----- % aberrations in aetaphase ---------- % aberrations in posimetupha.se 120 100 80 a, 40 20 0 15 30 Time (minute) 45 60 Figure 1. Quarterly Variation of Mitotic Indices and iiberrations in Various Stages in Control Runs of Onion 120 100 30 So Time (minutes) Figure 2. Quar.ter^y Variation of Mitotic Indices, and Aberrations in Vuriaus Stages in Treated Runs of Onion o o> co Fh o o- X3 o oo CO •H S LQ CO 0v2 o o\i c o o 93p?q.U30J8cT o to o o CSZ Variation of Mitotic Indices and Aberrations in Various Stages in Continuous Control of Onion Oi Figure 5. Hourly 17 XI rH O CO o Oi O -P ao ■H -U> -p -P CO s ^ v< tM u} CO —I r-f o o CM rH o o o CO 0.^T3q.U9DJT0J o CO o o Figure 4. Hourly Variations of Mitotic Incdces and Aberrations in Various btages in Continuous Treatment of Onion 18 ca a Tj +3 O C3 00 -C d o •£H a> CO O x> .Q £1 LQ CO 02 O 00 O C O o rH o o CO o SrTV^UOOJOrf o •4< O E-t Variation of Mitotic Indices and Aberrations in Various Stages in Runs of Onion to Figure 5. Hourly Recovery lb Deviation of propane % from control Deviation of metuphaue % from control — — Deviation of post-raetuphay e % from control +50 +20 a •oH -P aJ > a> -10 -20 0 15 30 45 60 Time (minute) Fifure 6. Quarterly Variation of Mitotic Formulae in Control Runs of Onion 430,- +20 +10 / c: o -P 0 > a, o ?-. "i, ■a H /' a d o rH \ O H o u OJ y o u o y £3 d o o •H *H Sj <3 aJ •H -H > O > Q Q « / d •H <» d i/ "O -p -P -P -• id Cl S-4 o C«4 o -p o -p K\ •H N d O o J) / / o •H Sh cd i - ,\i \ .' !-> u d o 33 . ■>/ \F o o + + i/3 o to o C\2 o H- UOT'^'RTAQa o S> rH \ vi •H -P o o o Cvi 1 o cO f 3) 2 i \ rH O Sh a d o rH o o o H -p a aS> Cj q c! o n a aS< o o a ’fcSt. a o o 03 t■ -H 03 'Fa-t ^ s\ "a a) A J3 3 Tt, T a, a j -p o -P 03 u 03 O a« a \ \ oo \ at at o o o C! eS fi at O o *H •H a> -P 3 •H •H > > 03 0) 02 \ ^ a, / o 03 Q Ct Q \ \ / \ - lO \ N / / \ X / \ \ \ :\2 V / \ ✓\ t s' k -i o + o tO + o 02 + o r~i + o O _L O o 02 rH 1 \ U0Tq.T?TA9a O to 1 o I o tQ I Variation of Mitotic Formulae in Continuous Treatments of Onion tO i\ Figure d. Hourly \ *3 \ \ O Ct -p +3 d S3 o O CJ o \ / / o fH p f ol o ( a o p p ^ V \ / a^ m o o c, 0 t-tvi ua / \ -•>} a, 'sA d 0 LtJ 51 4) 0 <13 53 -P /\ ! \ d d a d o, > o / !/ o« d p 00 o /\ O -p 03 fn t> > 0) CD CD QQO :i i / y CO / \ / V lO p p \ \ p Cf 0 a p d 0 f t I \ \y tH o \ P \ o d \ v o + d* CV .p. ik o LO *H t>- P \ o o o JO + o c\i + o p -4- UOT^T?TA0Q o I —1 I o C\2 I o o I Figure 10. Hourly Variation of Mitotic Formulae in Recovery Runs of Onion rH O JO <±4 -— Mitotic indices as % of control % aberrations in prophase % aberrations in metapnase % aberrations in post-metaphswe 100 a) SO 80 c sJ -P f al > o U 60 (D u, 40 0 1 Z 3 4 5 Time(hour) 6 7 8 Figure 11. Hourly Variation of Mitotic Indices and aberrations in Various Stages in Continuous Control of Pea 100 a> CL, 40 Time(hour) Figure 12-. .Hourly Variation- of alltatic Jndie.es. and AberraiJons. .in Various Stages, in Continuous Ireatiaant .al‘ Pea ----- Deviation of prophase % from control Deviation of metaphase % from control Deviation of post-metaphase % from control +■20 +10 fi o • H -P dJ •P > CD Q \ \ -10 —2 0 __ 0 i_ 5 4 5 Time (hour) Figure 15. Hourly Variation of Mitotic Formulae in Continuous Control of Pea lime (nour) Figure 14. Hourly Variation of Mitotic Formulae in Continuous Treat­ ment of Pea F6 rH M CD O jj a) © no a, mo 1 ^ rH r 03 CD 1 -P H 'O H .3 *H -P © CD S 1 —1 -0 © 4 O, S a. rH o a) a) ri 23 Figure 15. Ontogeny of Mitotic Deviations Inclucod by Lindane ex, Cl) co £ 41© u <4 © M3 id a, mi 0, g © H O 3 t—? 43 rH H i—I O C_3 4 j E-4 -4 © o 4 r> -o JH 4 M) 43 Cl) r j 4 no fn no ,4 4 i-4 rH CO O q *=3 Q 4? §> © cd 2 © CO 4 .d 43 Q) 41 y-s +3 43 -31 P © Hi CD M3 43 4 o a ) s c-t d-4 n no 43 no 4 *-4 CD CD CO >._s © cd a) CD CO -P Hi Hi Cd CD TJ 4 rH CO CD ojD o .d a, bj) O Xi g 3 u 4 M) O <£» © 41 CQ ^ C/l O, CO Spread Prophase (Segregated aj d 3* 9 ’-4 ara •d rH 1 t rH rH •H © 'I © 43 o 43 Ci oj © © © •agj 4> a H no O " ©1 4 d r i— 1 H a: ■rf o a. PQ 3 21 © at Hu C -C 0 ,4 1 41 M q © H cu 43 4 © g d no EH 3 4 M3 ci H Hi O HI 3j ----rH o a, © CO Hr 3o, 1 © rH *H 4 03 O d Hi 4d1 ai. O 04 4 aJ 4 4 3! r-t 27 II. Aberrations For the purpose of comparison, normal mitosis for both onion ana pea is; shown in Plate I, Figs. lb, 14, 15, 16 and Plate IV. In general the normal sequence has been intensively studied and well established. The description can be obtained in any cytological or genetic text book. However, it is worthwhile to stress one often overlooked point here, that is, after the breaking down of the nuclear membrane at the end of prophase, the chromosomes clump in center of the cell ana form a more or less solid ball with the nucleolus or nucleoli included. The chromosome ball may or may not go through a brief pause, then the chromosomes in the tangled mass start to line up on the equatorial plate. This untangling action and line-up movement may appear to be rather chaotic before all the chromosomes are arranged on the rnetaphase plate. is designated as prometaphase. This interphase stage It is rather infrequently seen in normal onion root-tips, and tnis may be attributed to its relatively short dura­ tion. However, there is no doubt that this series of events is normal for onion though not as clear cut as in peas in which 5-9 percent of the dividing cells in normal root-tips may be classified as prometaphase. The entire process of mitosis involves continuous change, thus inter­ stages and %'wkward "figures can not be avoided in a sudden stop by fixation. Thus the abnormalities or aberrations are scored in Cl relative eense. Criteria used for classification of aberrations are degree of chromosomal contraction, arrangement or relationship of chromosomes or chromatids,' direction of chromosome or chromatid movement or degree of spindle £3 inhibition, end finally .reversion of chromosomes or chrometio:.All the criteria arc ssel 1-expianatory, except the degree ot chromo£omal contraction v.'nieh needs, soi/ie further explanation. The normal size is the size v.hich can be found ir\ the corresponding stage of untrea tecL material. Chromosomes are referred to as overcontracted v/hen they are much shorter and denser than usual for the stage concerned. For instance, in onion matericiL the over-contracted metaphase chromosomes were only approximately one third of their normal length. The classification ana occurrence of aberrations may be aescribed as follows. 1* Prophases There are three kinos of detectable aberrations in prophases; namely, spread* segregated, and overcoritracted. The spread prophases are composed of contracted prophase chromosomes lying randomly intide a nuclear membrane which is more expanded than normally, thus they give a spread appearance (Plate I, Fig. 1; Plate XI, Fig. 1, Plate V, Fig. £)• Segregated prophases, often referred to as "reductional groupings”, are aggregated into two or more groups. The nuclear membrane may or may not be visible depending upon the developmental stage of the cell (Plate I, Fig. 1; Plate II, Fig. 1). Over contracted prophases are cells with over- contracted chromosomes arranged along the nuclear membrane or in a definite sphere (plate I, Figr. 4). The spread condition is a stage in tne normal sequence of mitosis, but its duration is short and its appearance seldom exceeds five percent 2i) in a roct-tip. pea. Treatment rc.ic.eti its incidence grcctly both in onion and This condition was couu.ion in shorter ti*ie oi' treatment anu ha;. never seen iounci 1l.-.ter tnan two nours recovery alter one hour treatment ot onion (Table 5). The segregated propJha.se has been observed in onion controls but not ircquently. In this investigation, it occured rather sporadically both in treatment and recovery, the highest point being reacned alter 24 hours treatment* So iur the overcontracted cases have not been observed in controls, ana were rare in treatment and nave only been bound alter 46 minutes treatment ol onion and ei&nt nour treatment ol pea* None oi these prophase abnormalities showed any very definite trend* 2* Metaphase: All the metaphase aberrations may be classified into seven categories: (1) The least abnormal one is the disorganized metaphase which consists of more or less normal sized chromosomes but without definite orientation or disturbed orientation* Occasionally this type of abnormality appeared in control materials, but was common witnin one hour treatment ol onion, though sporadic in recovered materials after one hour treatment oi onion and continuous treatment of pea. {2) Spread metaphase: This is characterized by contracted chromosomes in ranaom arrangement* (Plate I, Figure 5). The hignest frequency of this kind of abnormality was reached at one hour treatment in continuous treatment ol onion and two hour lor pea, then gradually tapered off as treatment vras prolonged. Tnere were two peaks in recovery runs of onion, 30 i.e. at one hour treatment ana eight hour recovery alter treatment. This variation was uncommon in controls but did occur. (3) Scattered metaphase: This deviation is characterized by over­ contracted chromosomes, or so—called diplo—chromosomes, which are scattered all over the cell (Plate I, Fig. o; Plate V, Figure 3). c—metaphase ana has not been observed in control tissue. This is. the classic The Frequencies oi the scattered metaphase increased with time oi treatment in both continuous treatments oF onion and pea. In recovered materials of one hour treatment oF onion, the peak was reached at two hours alter treat­ ment ana gradually tapered oil. At eleven hours-, tne scattered metaphases, appeared again but were much less Frequent (4) Segregated metaphase: (Table b)• Separations into groups may occurin nuclei in wnich the chromosome organization is otherwise normal, spread or scattered. The chromosomes may be normal, contracted or overcontracted depending upon the degree oF the eFFect (Plate I, Fig. 2, Plate II, Fig. 2). This abnormal condition appeared sporadically both in treatment ^nd recovery (Tables 5, a, b and 6). Segregation oF normal metaphase occasionally occured in onion and pea controls. (5) Trie clumped condition; The chromosomes may be normal, contracted or overcontracted, ana tney are arranged in such a way that stickiness and clumping oF chromosomes are obvious (Plate V, Fig. 1). This type of aberration was very common in continuous treatment oF pea (Table 6), iujVcvp; ,nfc In continuous treatment of onion (Table 3 and 4), and rather inconspicuous in recovery runs (Table 3). materials• None has been round in control 31 (6) The ball type metaphase; In this type oi' aoerration, the chromo­ somes bana together to i'orm a solia ball, in which only mosomes can be observed. few Although the occurrence of this type in an earlier stage oi‘ mitosis is known as typical normal prometaphuse, it does not usually persist. It was also not especially persistent after ’'Lindane11 treatment as it is after colchicine treatment (Bowen 1B53, Hyypio un— public ned)* (7) The telomorphic metaphase: The chromosomes of this type revert to telophase appearance in metaphase condition. Such a condition has never been found in control materials of either onion or pea, but is fairly common in colchicine treated animal tissue. Such reversion was not common in most cases but did appear in one onion run after one hour recovery following one hour’s treatment (Table 5). 3. Post-metaphase: All the post-metaphase aberrations so far observed may be put into five classes s (1) The mildest effect on post-metaphase is shown by disorganized figures, in wnich the normal arrangement of chromatids are somewhat distorted, altnough their shape and size are not aiHerent from control ones. root-tips. This abnormality occasionally can be observed in untreated Tnis is a common- aberration early in treatment ol both pea and onion (Tables 3, 4, 5 and 6). (£) The polar post-metaphase: The typical chromatics of this type of aberration are more or less normal in size and shape. Their arrangement 52 may be star—bipolar cr multipolar,,the iormer appears at two equal sized "stars" located more or less in the center of the cell (Plate II, Fig. 4, 5 and 6), the fatter is composed of three or more unequal "stars" scattered aoout the miuule ol the cell (plate II,Fig. 7 ana 8). Similar figures have been found in controls- but with very low frequency* In this investigation, the hignest frequency was reached after one hour of con­ tinuous treatment of onion (Table 5 and 4) ana two hours for pea after which it became gradually rare (Table 6)• These atar-anaphasee and telo­ phases were commonest in onion in the early recovery stages following one hour»s treatment (Table 5). (5) The apolar post-metaphase: This is the most common effect of continuous treatment and the frequency increased with time (Tables 4 and 6)* In recoveries, no definite trend of any sort was apparent except that it appeared quite frequently (Table 5). The so-called apolar arrangement is a condition in which the overcontracted, bar-like chromatids cio not chow any direction of movement and lie loosely or clumped in one group at random in the cell. (Plate I, Fig. 7; Plate V, fig. 4). If the chromatids show any sign of reductional grouping, then it may be cubclassified into the following £,roup. (4) The apolar segregated post-metaphase (Plate I, Fig* 3): This was found mostly auring continuous treatment of pee ; this type of deviation showed a more or less increasing trend with time (Table 6). (5) The clumped post-metapnase: or apolar segregated This is not a clear cut multipolar post—metaphase, but is characterizea by stickiness 35 oi clu omatias wia groupin^c. This type ol' abnormality shosed no definite tx end and wat- pare in tree.ted pea roots in all rune (Tables 4, 5 and 6). The last mentioned three types oi aberrations have never been iound in control materials, ana are more cnsrscteristic oi colchicine treated materials• 4. Heating stage: Aberrant resting stages may be classified in live groups^ namely^ polyploid uninucleate, binucleate, multinucleate, fragmental nucleate, and pycnotic. The uninucleate cells have been found after two hours treatment in both onion and pea (Tables 4 and 6)• Binucleate cells occured as early as 15 minutes in treated onion materials, but gradually decreased in number in longer treatments. Multinucleate cells were more frequently seen in both treated and recovered materials. Some workers used sticky, lobed, dumbbell, ring, etc., to describe the appearance, as a whole, the fragmental-nuclei resemble tnat of leukocytes in blood. Only in long time treatment (about £4 hours) or treatment of dinner concentration (more than J.%), pycnotic nuclei have been observed; occasionally, some patches of cells appeared pycnotic in less affected and even untreated materials, this may not be considered as a result oi treatment but rather as oi unknown nature. III. Comparison of Results In summarising the results obtained from this investigation, several remarks seem worth stressing both for theoretical and practical reasons. 34 1. Comparison oi Onion and Pea; Generally speaking, the pea seedlings provide better materiel lor ex­ perimental cytology ol the type reported herein. It is simply because the mitotic indices are higher ana mitotic aberrations lower in untreated pea than that in onion (Tables^ and Bowen (1955). tnis the writer agrees with The standard errors of the means ol mitotic indices for pea and onion are almost identical (pea ±.259, onion ±.287, first row of Table 1 and 2). So individual variability is about tne same. The fluctuation of the mitotic formula is similar in both cases (first row of Tables 1 and 2). The irequencies of metaphase in onion controls are more stable* and the frequencies of post-metaphase in untreated pea, on the other hand, are less variable. As for reaction to treatment (Tables 3, 4 and 6), the onion meristemic cells are affected more quickly and uniformly, while the pea root-tips showed maximum abnormality at three hours later during the treatment. D* Amo to (1949) reported that onion is more sensitive to Y-hexachloro- cyclohexane than pea, however the nutrient solutioxi used in experiment may lessen the effect to a certain degree. The kinds of aberrations are similar in both materials, except that clumped metaphase is much more frequent in pea than in onion* Presumably the clumped metaphase is derived from clumped prometaphasej since the tightly packed type of prometaphase is more frequent in control pea, it is not unexpected that there would be more clumped metaphase in treated pea* In addition, the size of the meristemic cells of pea are far smaller than that ol onion, 6b some less spread or scattered figures may be easily lumped into clumped condition. The clumped post-metaphase is a ratrier common eil'ect in onion but not in pea, this may be explained by tine fact tnat onion is more sensitive tnan pea to this particular insecticide. Comparison ol Control, Treated and Recovered Materials; The control materials are rather normal and can be used as standard lor comparison. There are tnree definite types oi aberrations in treated material (Tables 3, 4 and 6) i.e., spread prophase, scattered metaphase and apolar post-metaphase. The first type of abnormality does not have any trend, but the last two kinds definitely increase with time* Other aberrations described in the second part of this chapter may be considered as either intermediate between control and treated cells, such as disorganized figures, or as sub-groups of the above mentioned three distinct aberra­ tions, e. g., apolar segregated anaphases. Some segregated polyploid figures observed at £4 hours treatment both in onion and pea may not be considered as sub-group of any simple aberration, since their origin is not due to chance alone but lie in binucleate or multinueleGte cells which are formed in treatment of the previous mitotic cycle. In recovered materials after one hour treatment (Table 5), no definite trend of aberrations can be observed, spread metaphase and polar post­ metaphase are ratuer common abnormalities. originates from relatively noxmial prophase. Probably, spread metaphase Polar post—metaphase indicates 36 partial inhibition oi spindle activity, and also may be a sign of re­ covery after complete destruction of spindle function (Levan 1958)• Since lfLindanen is insoluble in water, recovery is relatively difficultj&nd the characteristic effects, scattered metaphase and apolar post-metaphase still continue to appear sporadically presumably because of residual traces of the chemical in the tissue. IV. The Abnormal Mitosis Induced by Technical Lindane Depending upon the mitotic stage of the cell at the time the technical Lindane became effective upon it, there are four possible ways to complete the mitosis. The first one is that the complete mitotic cycle proceeds under the influence of the chemical, the last three will be partially affected by the chemical. The procedures are illustrated in Figure IE. The parenthetical names are possible subgroups. In any treated root-tips, these four types of abnormal mitosis can be observed simultaneously j however, the first type will be predominent in longer treatments. 37 TABLE 3 PERCENT DISTRIBUTION QE ABERRATIONS IN MITOTIC. STAGES IN SHORT TIME TREATMENT. OE ONION Prophase »-3 tr a> % of Total •-a o c+ Metaphase S3 O P. P.. Co <0 P p i CO O CD < C*, c+ at hi hi hi at P I o p 4 p. ss — 28 60 14 14 4 8 12 24 36" 13 33 11 8 — 20 30 — 35 20 15 9 22 — — 45 33 3 38 TABLE 4 PERCENT DISTRIBUTION OP' ABERRATIONS IN MITOTIC STAGES IN CONTINUOUS TREATMENT OF ONION t-3 g* CD tr *-i Prophase % ol Total >—3 H O OI c+ S2 to to c + O CD to CJS E p. »= CD CD P- P CL Metaphase % of Total £2 O § P. OS P to HCQ O 4 OS to to to CD OS to o P CD OS c+ c+ CD 4 CD •-i CD to P c+- b Htsi c+ CD CL CD to CD XL°^.t-rtie^t.aphase _ % of Tatal »-3 O c+ tr & p to H* W o CD os P. Vi o a 98 2 — 13 23 77 — — 30 j40 'i 54 98 2 35 85 6 6 z 44 96 2 38 77 5 18 tr 3 — to CD to CD OS 10 — 13 — 37 18 18 — 36 9 57 17 17 — 17 — j 11 CD * ? 1 P > to t0o o a-' H P P 1 H* N CD CP to — — o M P P to 57 to — — |l3 1— - — \ 3 36 97 3 47 81 4 35 97 3 37 73 5 33 97 43 I— i 63 4 — 2 15 — — )17 ‘ ----- — 6 77 27 — — 28 ------- — 7 79 7 7 — 24 — . 88 8 4 25 76 16 8 90 — 33 2 — J j— 60 — 40 — — 70 — 30 — — 119 ,----- 6 33 100 — 42 7 41 98 — 40 I— 8 15 93 — 46 9 23 100 — 31 I— 52 10 38 100 — 37 42 j— 26 4 — 9 ' — 67 3 — •39 45 3 — ;46 1- — 46 3 — I•y25 ----- \lQ I J30 |- — 68 6 26 — 76 7 17 — — 88 12 — — 100 — — — 83 17 — - 77 23 — I i — 34 23 97 3 — 40 25 41 35 24 57 - 11 48 100 — 14 30 24 17 - — 3 — 77 — — 72 — — 88 — 12 26 i 10 - - — 39 TAbLE 5 PERCENT DISTRIBUTION OP ABERRATIONS IN MITOTIC STAGES RECOVERY RUN OP ONION HI £ Metaphase 3 3 28 — — 17 70 10 3 52 — — 38 — — 24 55 3 18 55 22 5 — 37 — — 45 49 6 — 7 51 35 7 so — — 20 30 5 45 — — 80 12 8 — 27 — — 26 30 3 41 22 10 — 85 5 — — 16 — 31 25 — 44 24 — 8 72 16 4 — 22 — 82 18 — — -- 30 — — — 25 4 4 88 — 4 4 30 — 3 94 — 3 100 — — 25 — 4 88 4 4 1 3 \ 27 100 — — 43 3 3 73 21 — [30 49 100 — — 26 8 — 78 14 — 25 16 L—- —« 91 9 — 39 — 2 41 95 5 — 21 — — 48 3 41 100 — — 22 — 18 4 53 98 — 2 27 — 5 48 98 — 2 25 6 62 100 — 7 54 98 8 54 100 — 9 51 100 — 10 i 45 96 11 48 23 — —, — 2 *o ►i 05 P P- r o •2 5 o — ■ i 33 !» Disorganized Segregated l Spread t *-3 (D Clumped Apolar-seg. —— 61 i 33 5S o ; o to Apolar % of Total Disorganized u c+ fa M Vi jNormal % of Total in Segregated •-3 H3 O - % of Total tz 0 sf I-* Vi Vi 1 H1 P q s t-metaphase Scattered tr ► •i Prophase 16 — — 50 37 — 13 24 8 — 34* 12 -- 46 — 25 — — 56 44 — — — 127 — 15 55 — 50 — 13 51 — 30 — 16 40 — 28 100 — i t t 6 40 TABLE 6 PERCENT DISTRIBUTION OF iUiERRATIONS IN MITUTIC STAGES c o n t i n u o u s treatment ,o f Time (hr.) Prophase % of Total is o M *o 3 P CO CD 0«S 4 CD CVj P> c+ CD P- Metaphase J6 of Total 1-3 O 0 H 1 o o P O c+ & P ea . IS O H H fls (—1 c+ 2 o t? H* tfc O 4 ov, *. P H» ESI CD p CO *C3 CD (t ptl CO o *» c+ c+ CD 4 CD & cn CD 0's CD crc, P c+ CD P O W 2 *o CD P tr P! M H 1-3 O c+ P Post-metaphase % of Total !2 O 4 o H* !> o to O M 4 ►j *D O P H P 4 1 to CD OS* P 0*5 -P** bs • CD P- C+- > o *o CD P 1 66 88 12 — — 15 53 7 20 7 — 2 62 84 16 ■— — 25 13 4 52 9 13 3 51 84 16 — — 30 3 3 36 33 3 4 50 88 12 — — 31 — 22 29. 6 45 76 14 — — 39 — 2 31 18 26 20 a 53 94 4 — 2 31 — 3 32 32 7 23 24 42 81 2 17 40 — 5 60 23 12 — —- — 13 — 19 68 11 11 9 — 15 34 7 13 13 33 m — 19 26 — 5 37 32 29 r— 19 — -rr' 5 68 27 3 16 — - T “ 6 44 50 3 16 — -- 6 63 31 18 ___ 5 5 50 50 41 bILCUSlION I. Mitotic Aspect One immediate eiiect oi' this insecticide on mitotic meristemic cells is the complete suppression oi’ cytokinesis. So long as treatment is continued, no cell will divine regardless oi nuclear organization* Even in recovered materials alter treatment oi one hour, no cell plate has been noted belore 48 hours. That might be attributed to the lact that ( a the chemical is practically insoluble in water, so cioes not readily leach out within relatively short times, and (£) it takes part in cellular metabolic process and is utilized or translocated in the tissue. A binucleate cell is capable oi undergoing a second mitosis which is normal ii treatment is stopped. In the case oi continuous treatment, each nucleus will travel any path Illustrated in Figure It, thus numerous complications result, Figures 1 - 6 in Plate III, Figures 5, 6, 7 in Plate V, demonstrate some oi these* polar anaphases originate Irom normal early anaphase or metaphase as illustrated in Figure lb. Multipolar anaphases probably arise irom partial breakdown oi the spinole mechanism. diiiicult to explain by spindle malfunction. "Star” bipolar anaphase is In the short time treatment ol onion, some intermediate stages between normal early anaphase and "star1* bipolar or multipolar anaphase have been observed (Figures 4, b and 7 Plate II). The configuration and relatively abundant occurence of these cells led the writer to believe that there are cielinite attractions 42 ctiuong anaphase kinetocnores uiter tue "spinule mechanism" hat been inhibited partially by any chemical or physical meant. The spatial re­ lationship is ratner a determining factor in grouping, since "segregetional groupings11 are more or lens random. Gaulaon ana Carlson (Idol) stated thatt Observations on living neu.roclasts reveal that stars (metaphases) can be formed in two ways: First, they may appear daring either partial or complete destruc­ tion of a fully formed spindle boay by strong concentra­ tion of colchicine. Second, enough spindle material will be organized at prometaphase to form a focal point of a star. Thus we see why Barber ana Callan observed stars most frequently during the first hours of exposure to a relative strong solution, ana wriy Levan and Peter observed tnem most frequently during recovery when the intracellular concentration of the alxaloid was being diluted. They also sensed the attraction of metaphase xinetochores which may be present in partial inhibition of spindle either on the way to total breakdown or recovery. Tne above mentioned facts further illustrated our notion (Wilson Hawthorne and Tsou>19bl) that the spindle action in metaphase and ana­ phase can not be adequately defined in terms of a single force, but that at least two more or less independent forces are involved * first, the forces between kinetochores and so-called spindle "poles1** and second* the affinity among kinetochores. The kinetochore attraction is probably partly responsible for metaphase "line-up" and anaphase migration in normal cell division. sidered as too far Laggard chromosomes or chromaticis could be con­ separated to be attracted, or are in equilibrium 4o with two or more attractive forces initiated from aojecent groups; a thira possibility is that tne Kiiietochore of laggaras is partially in­ activated by the chemicals (Daniel and Wilson, in press)* The cleavage oi the kinetochore seems to be a unique jjrocess regard­ less oi degree ana kind oi effect. Sometimes it may delayed as in the case of acti-dione ana colchicine (Hawthorne and Wilson,1952; Bowen, 1955). Omission of this process is rather rare in treated plant tissue, thus Levan (1954) claimed that the difference between colchicine effect in plants and animals is that there is usually reversion from metaphase in the latter* The scattered metaphase is likely the result of complete inhibition of both "kinetochore-poles" and*kinetochore-kinetochore" forces, so that tne "diplo-chromosomes" or "c-pairs" remain in the arrangement of prophase or?in most instances, may ’'explode1' a little under the influence of a diakinetic like force. Segregated figures of this scattered type showed no definite orientation, so that it may be assumed that segregation is partly due to chance, and partly due to attraction of Kinetochores. Apolar postmetaphase is just the continuation of this type of abnormal metaphase. Spread prophase caused directly by chromosomal contraction appears to be a more or less immediate effect of technical Lindane on onion meristemic cells (Table 5, Fig. 1 in Plate II; Fig. 1 in Plate 1). Over- contraction of chromosomes seems to be a common eliect of any chemical, from the simplest ethanol (Vaarama,1947) to heavy metal salts (Macfarlane, 1953), alkaloids (colchicine) nucleates (Powell, 1952) and antibiotics 44 (Mlson, 1950; Wilson unu Bowen, 1951; Bowen and Wilson, 1954). It is unlikely that chromosome contraction can be explained by simple ueiiydr&tion as figenaga (1949) proposed, since some effective concentrations.were too low to cause a nypertonic conaition.lt,as in many other biological phenomer h „ is a result of complicated bio-physico—chemical interactions, involving surface actions on the matrix and degree oi chromonema spiralization. The relatively frequent appearance of segregated prophases in short time treatment of onion suggested that "segregational groupings" or "reductional groupings" can not be defined as splitting of the spindle into several parts, but is ratner due to chromosome contraction plus relative expansion of the nuclear membrane and possibly inter-linetochore forces. As for metaphase and anaphase, the segregated figures have been discussed above and it has been suggested tnat chromosomal autonomy and chance also play some important role in the initiation of these aberrations. II, Chromosomal fragmentation Isolated chromatins and chromosomal fragments have been observed in angiosperm seedlings after direct treatment with tne insecticiaal powder (Kostoff, 1949). DfAmato (1950) treated onion root-tips with dilute ethanol solution of pure gammexane and found a frequency of chromosomal fragments in 150—160 anaphase figures obtained from three root— tij-'S of ter three days recovery of approximately 55 percent. The writer nas analyzed more than tiiirtjf seiies of onion te^t,., with each series containing about tnlrty root—tips. The irequency oi true 45 ir.ee chromosomal fragments was only one ior each ol six treated root-ti^s. On the otner nanu, tour cases also have been iouna in untreated materials. Attached fragment! were more frequently observed in do tin treated ana un­ treated pea root-tips; tnis may be attributed to the entanglement oi tne four secondary constrictions of chromomosmee in pea* Levan, and Tjio (1548) found as high as sixty percent of phenol treated cells containing fragments* Spontaneous chromosome fragmentation has also been detected in Vicia faba (Levan and Lotfy, 1550). Moreover radiomimetic activity has been demonstrated for chlorine in meristemic cells of Pisum rootlets (Von Rosen, 1958). In view of conflicting results concerning tne potential raaiomimetic effects of Linuane, further investi­ gation seem desirable. III. Polyploidizing Effect Nybom and Kmitsson (1947) claimed that Linaane is a better polyploi­ dizing agent than acenaphthene and only second to colchicine* The writer did several experiments on water soaked rye (Secale cereale L*), radish (Raphanus sativus L.) and pea seeds. The majority oi meristemic cells became polyploid witnin 48 nours treatment v/itn 0.01 to 0.5 percent suspension of the technical Linaane. Selected polypLoid seedlings have been planted in green house or fielu, but, to ante, only diploia parts have grown out. Soaking seeds directly in a suspension ol the insecti-ide also was used as a means of inducing polyploidy, but none ol tne emeigent seedlings grew to maturity. 46 With colchicine one may expect successful recovery of polyploiay once in several hundred treatments. With Linaane tne percentage of success is obviously much less. IV* Inositol Antagonism According to Kirxwood and Phillips (1946), the growth inhibition of Saccaromyces cerevisiae by 'tf-hexachlorocyclohexane can be overcome by a sufficient amount of m-inositol. Buston, Jacobs and Goldstein (1946) reported that m-inositol is an antagonist of Lindane on Nematospora gossypii in some growth experiments. In addition, Churgaff, Stewart and Magasanik (1948) claimed that 11.. .meso-inosital is able to inhibit the metaphase arrest and tumor formation induced in Allium cepa by colchicine or gammexane.”. On the contrary, Schopfer, Posternak and Boss (19a7) dia not succeed in demonstrating an antigammexane action of m—inositol in older species of Saccaromyces, and Eremothecium blakesleanus and Ustilago vlolacea. aahbyii. Candida spp., Phycornyces Failure of m-inositol to protect sea urchin eggs from the toxic action of S'and Y-hexachlorocyclohexuue has been also reported by Chaix and Lacroix (1943)• In between, D*Amato (1949) considered that m-inositol delayed the effect of gammexane on onion root—tipsin a similar manner as sugar \ solution, which merely changed cell permeablity. These conflicting reports led the writex' to re—investigate. Exact repetition of C hargaff fs experiments failed to show any antagonism of 47 Lindane and in—inositol. As a matter of fact, in—inositol alone induced similar effects in onion dividing cells as those induced by Lindane* Further experiments, using equal parts of technical Lindane and m-inositol of comparable concentration on pea seedlings, gave the same result. The slides made from the first four hours treatment as well as after twentyfour hours treatment demonstrated perfect Lindane effect — "c-mitosis". Woolley (1952) explained the antagonism of m-inositol and f-hexachloroeycl-ohexane or Linaane in terms of solubility and configuration, i.e, the former enters tne cell tarough the aqueous phase of the cytoplasmic membrane, tne latter the lipid phase; since both chemicals have the same 1I configuration, antagonism arises. According to Ostergren and Levan (1945), c-mitotic activity is a kina of narcotic effect. Hober (1945) says; Typically, narcotics do not enter into a chemical reaction with cell components; they are cnemically "indifferent11. The reactions they enter into with cells are of physico­ chemical rather than of chemical nature. Tney make contact with cells by seconaary valences, changing the surface properties of exterior or interior cellular structures and microstructures, wnich become apparent as changes of aispersity, of hydration, of colloaial aggregation, of dissolv­ ing power, of absorption affinity. Therefore inositol antagonism may be found only in inositol-requiring organisms, but not in others. V. Difference Between Lindane and Colcnicine Ever since Ostergren and Levan (1943) used hexachlorocyclohexane to study c-mitotic activity, it has been recognized that this chemical 43 and colchicine belong to the came category as tar as their mitotic efiects, inanetion ol polyploidy and c—tumour are concerned. Ostergren, (1945) stated that both chemicals caase narcotized mitosis and proposed a precipitation hypothesis oi‘ narcosis to explain the mechanism of c— mitosis* Levan (l'9ol) classified the effect of both chemicals as reversible physiological reactions, but”*..colchicine differs from the substances (include Lindane) of simple narcotic effect in so far as c-mitotic activity is extended far down into concentrations which are at a great distance from the saturation point.” Judging by the results that have been obtained by Bowen (1955) and Hyypio (unpublished), Lindane and colchicine differ in degree but not in kind of effect on mitotic activity- For instance, ball metaphase is a prominent aberration after colchicine treatment, but relatively rare in the case of Lindane. It may be attributed to the fact that colchicine immobolizes prometaphase chromosomes whereas Lindane affected chromosomes still are capable of moving out of the prometaphase mass; but due to in­ hibition of spindle function and lcinetochore attraction, the arrange­ ment of chromosomes is completely destroyed. According to Powell (1951), various nucleates show effects at late prophase, with colchicine primary effects are at mataphase* The results obtained in this investigation suggest that Lindane exerts an initial effect at prophase which is revealed at late metaphases. This suggestion also explains why spread prophases and metaphuse can persist in continuous treatments; and why Lindane affected chromosomes are capable 49 of moving out from prophase. Tne quantitative disagreements are: First, Lindane is not as potent & mitotic stimulator (Table 1 and 2) as colchicine as Bov/en concluded (1955.). This point agrees with the result of Scholes (1953). Second, Lindane does not block metaphase to anaphase movement to such a degree or for so long a time as Hindmarsh (1951) showed in her colchicine treated material. (Tables 3, 4 and 6 post-metaphase frequency decreased only within one hour, after that the percentages remained essentially the same)• Third, the frequency of prophase is relatively reduced in Lindane treated materials (Tables 1, 2, 4 and 6). This may be attributed to shorter duration of prophases or to a relatively increasing number of metaphase-like figures which without chromosomal contraction and absence of nuclear membrane will be classified as prophase. This suggests that the nuclear membrane may break down in a rather shorter time than normally. As was suggested by Huston (1952) when she studied the cytological effect of the fungicide, Thiolutin, there is a correlation between "over­ con traction1' and increase in the relative number of metaphases. All this may indicate another possibility that the total division cycle is somewhat lengthened as Guttman (1952) noted in colchicine treated material, but not in the same proportion to that of a normal one or of a colchicine affected one. In 24 hour treated pea root-tip, octoploid cells were detected. According to Brown (1951), the mitotic cycle of pea root meristeraic cells is about nineteen hours. Thus the cycle is definitely shortened 50 by the chemical. Bowen found the same effect in colchicine treated pea root— tips (1955)• Whether the duration of mitosis or interphase is being shortened is difficult to determine from fixed material. . VI. Explanation of Phyto-responses Growth has been separated traditionally into two rather distinct phases, cell division and cell enlargement. The former involves the mechaiiism of cell division and chemical reactions of protoplasm synthesis, the latter with growth of cell wall, possibly some bio-synthesis, and the physical process of hydration or vacuolation. The superficial cytological course of cell division and cell elongation have proved to be rather similar in roots and shoots, although they are different with respect to their physiological behavior. The mechanism of cell division is still not well known. However, the insecticide affects the normal cell division as observed by the writer and many other workers. The products of the abnormal mitosis are polyploid, binucleate, and multinucleate cells and fragmental nuclei. In subsequent cell division, the tetraploid becomes octoploid in continuous and residue treatment. The binucleate and multinucleate cells, under in­ fluence of the chemical,, may divide and form polyploid cells with higher or lower number of nuclei, depending upon the distance between nuclei. If the chemical is completely leached out, cell plates may form and aneuploid microcytes are formed. The fragmental nuclei react sometimes as polyploid cells other times as multinucleate, largely depending on the manner of 51 grouping, Levan and Ostergren (1943) stated that an increase in the number of chromosomes in plants results in slower growth and lower growth energy. Aneuploid cells tend to be less viable, and Kostoff (1949) thought that they may become dead cells in tissues. Reduced cells or microcytes generally posses reduced viability. As for protoplasm synthesis, Chao and Loomis (1947) stated tnat hormones were formed as a by-product of protein synthesis; while Baldovinos's (1953) results suggested that protein synthesis was dependent upon hormones in non—green tissue. Nevertheless, some correlation between growth hormones, protein synthesis and cell division seems to be present. Burstrom (1951) stated that; "Cell elongation proceeds in two phases, the first phase involves an increasing elasticity of the wall, which has been explained on the basis of a loosening elasticity of the wall, which has been explained on the basis of a loosening of the joints between the micellae.” Some protoplasm synthesis occurs at this stage in order to keep pace with enlargment of the cell. "The second phase is characterized by a hardening of the wall, as evidenced by a decreasing elasticity. During the second phase there is, further, a very rapid supply of nutrients to the cell so that the osmotic concentration does not decrease despite the rapid increase in cell volume.” largely an absorption of water. over the whole cell surface. Increase in cell volume involves "Elongation does not take place uniformly Apical growth is a well known leature of many cells, and such differential growth is probably a wide spread phenomenon in ordinary tissues.” The mechanism of cell elongation whether 52 it is a hormone problem or a question of morphologic and metabolic changes m the cell is rather beyond our present Knowledge* However hormones are almost certainly involved. Hence we may say that hormones are likely responsible for the direction of growth, and growth is the product of metabolism. Anything inter— ferring with hormone activity or metabolic processes, will disturb the precise, correlated growth. It is a well known fact that Lindane and colchicine induce swelling or c-tumour in the elongation zone of roots or stems of most plants, dstergren (1944) considered that c-mitosis and c-tumour effects are re­ lated but independent, and that the c-tumour effect is due to "...narcosis of the cell growth control*" induces Havas (1949) further stated that colchicine .deviation of the polarity of hormones* trans-location." It is probable that Lindane behaves in a similar way, and disturbs the dis­ tribution of hormones. In the course of this investigation, another interesting physiolo­ gical response has been noted. The onion root-tips after 72 hours treat­ ment with 0.1J6 technical Lindane suspension showed a much reduced meristem below the c-tumour, and sometimes protoxylem strands extended to the promeristem or elongation zone. A similar result was reported by Mac- farlane and Sehmock (1943) in colchicine affected onion root-tips. They believed this is an irreversible cytoplasmic effect of poisoning, which is comparable to the effect of Phenylmercuric nitrate on onion meristemic cells* Phenylmercuric nitrate is a respiratory poison which probably attacks the -SH groups of succinic dehydrogenase, lactic and gluco 53 dehydrogenases, cytochrome oxidase and catalase. In conclusion, they proposed that the colchicine and colchicine-like reactions are possible responses to enzymatic poisoning. All the metabolic processes involve mostly enzymatic activity, thus it is not improbable that prolonged treatment with colchicine or Lindane will inhibit metabolism and growth. In field contitions, at planting season, the amount of residual l i n d a n e " in the soil and the kind of seeds probably are the determining factors for germination of seeds. When colchicine was used for seed treatment, the following results were observed. stimulates germination. Low concentration More concentrated solutions delay germination, and sometimes, even inhibit. Bond (Croker and Barton, 1953) suggested that a concentration which was low enough to stimulate germination is still effective enough to induce polploidy. 2, 4-dichlorophenoxy-acetic acid has been reported to cause c- mitosis and c-tumour in onion roots (Croker, 1953). It also prevents oxygen from penetrating into barley seeds and thus inhibits aerobic germination (Croker and Barton, 1953). two respects. Lindane differs from 2,4-D in First, Lindane is known to cause narcotic effects on the cell; 2,4-D, on the other hand, reacts chemically with cellular components. Second, 2,4-D is a definite hormone-mimetic substance. Since they in­ duce similar cytological effect, there may be some correlation present. Hocking (1950) found that a mixture -of trichlorobenzen.es prepared by the break down of the alpha isomer of hexachlorocyclohexane caused plant 54 deformation and ultimately inhibition of germination with relatively small doses. In addition, Lindane dissociates a little in solution to give some chlorine ions. Chlorine is known to prevent germination of seeds (Croker and Barton, 1953). Even if the residue is not concentrated enough to inhibit germination, the .growth of emerged young roots and shoots is de­ finitely affected by physiological and cytological disturbances caused by the insecticide. The writer carried out a germenation test on pea seeas by the paper towel method. The germination percentages after three days for control and 1,000 ppm suspension were 99 and 97 respectively. The average lengths of shoot and root were 1 and 3.7 inches respectively for control seedlings, 0.7 and 3.0 inches for treated seedlings. After ten days treatment they increased to 7.8 and 6.0 inches for control seedlings, and 4.75-and 3.5 inches for treated seedlings. The control seedlings were straight and with numerous side rootsj on the other hand, the treated seedlings were twisted and with very few thickened side roots on stubby, brownish main roots. Root hairs are projecting tubes of epidermal cells, and chiefly function as absorption organs for water and mineral nutrients from the soil. The production of root hairs is believed to be due to hormone action, this may be the reason why there are no root hairs on "Lindane" affected plants. As for scorching of young leaves, the effect probably is partly due to physiological blocking of the carrier, and partly due to narcotic 55 influence of the chemical on the leaf tissue. The "off-flavored" effect is believed to be hue to chlorinated impurities present in the chemical. Since the pure chemical has been used for preparation of the commercial insecticide, no off-flavor has been reported. 56 5Jivii/ixiltY The re;-ulbs ootaiueu from this investigation could oe summarized at follows: 1. The 0*1% suspension oi' Linaane aia not stimulate mitosis ia root meristemic tissues oi' onion bulbs ana pea seedlings. 2. Unuer the ini'Xuence oi Linaane, the prophase frequencies in eiiviu- ing cells decreased than that of untreated materials. The metaphase frequencies increased signiticanti/, ana the post-metaphase frequencies remained essentially the same. Therefore, the "piling-up" of metaphase was probably due to shortening of prophase. 3. The immediate mitotic effects caused by Lindane were contraction of chromosomes; possibly break aown of nuclear membranej inhibition of cytoplasmic "spindle" mechanism; ana failure of cytokinesis. As treatment was prolonged, over-contraction of chromosomes, and inhibition of chromosomal attraction occured. 4. Inositol antagonism and radiomimetic effects of this insecticide have not been demonstx'ated in tnis investigation, thus further research is desirable. While Linaane is theoretically a good polyploiaizing agent, txhere are a number of practical difficulties which reduce its potentiality below that of colchicine. 5. The final products of atypical mitosis induced by tne insecticide weiepolyploidy cells and aneuploia microcytes. The former are gaint cells, but possess a much slower growth rate than that of normal ones. The 57 S e t t e r usually tend to be less viable. Therefore the treated meristemic tissues are much reduced as far as number of cells and rate of growth were concerned. 6. The insecticide also caused "c-tumour” in the enlargement zone of treated root-tips. Most workers believe that this effect is independent of "c-xaitosis" and probably is due to disturbance of hormone polarity at the cellular level. This disturbance causes abnormal growth, such as swelled shoot and root apex, twisted parts, lack of root hairs, distorted leaves etc. The precise, correlated normal physiological balance of some sensitive plants is definitely affected, when they are grown in soil containing the insecticide either as soil treatment or residue of spary. 58 BIBLIOGRAPHY Ashby, D. C., 1850, The Pi\yto toxic Effects of DDT. BHC, Panathion and Toxaphene. on Tobacco. Ann Appl. Biol. 57:624-639 Baldovinos, G., 1955. Growth of the Root Tip* Growth and Differentiation in plants. W. E* Loomis, Editor. The Iowa State College Press, 1S55. Boswell, V. R. , 1955.. Residues, Soils and Plants. of U.S.D.A.*284-297. Insects. 1952 Year Book Bowen, C.C., 1955. A Comparative Study of the Effects of Several Antimitotic s. Unpublished Ph* D. Thesis, Michigan. State College, 1955. — v anci G. B. Wilson, 1954^ A Comparison of the Effects of Several Antimitotic Agents. . Jour. Hered. 45:5-9. Brown, R* 1951*^- The Effects o f .Temperature on the Durations of the Different Stages of Cell Division in the Root Tip. Jour. Exp. Bot. 2:96-110. Burstrom, H. 1951. Mechanism of Cell Elongation. Plant Growth Sub­ stances. F. Skoog, Editor. University of Wisconsin Press, 1951. Buston, H. W., S, E. Jacobs, and A. Goldstein, 1946. logical Activity of Gammexane. Nature 1 5 8 :22. Cause of Physio­ Chao, M. D. and W. E. Loomis, 1947. Temperature Coefficients of Cell Enlargement. Bat. G&z. 109:225-231. Ch&iz, P. and L. Lacroix, 1948. Action des & e t 7f-hexachlorocyclo hexane sur l*oeng d 1oursin avant et apres la fecondatlon. Bioch. et Bioph. Acta 36-90. Chargaff, E., R.N. Stewart, B. Magasanik. Inhibition of Mitotic Poisoning Meso-inosltaL. Science 108:556-558. Crocker, W. and L. V. Barton, 1953. Physiology of Seeds. Botanica Company, Waltham, Mass., 1953. Chronica Croker, B. H*, 1955. Effects of 2.4-dichIorophenoxyacetic acid and 2 t4 T 5-trichloroohenoxyacetic acid on mitosis in Allium cepa. Bot. Gaz. 114(3):274-283. Cullinan, F. P., 1949. Some New Insecticides - Their Effect on Plants and Soils* Jour. Econ. Ent. 42(2);587-591. 59 D 1Amato, F., 1949. Sull|impiego Del Gammesano Come Agente Polipioi— (Ufa*Q ol gammexane as a polyploiaizing agent.) . Caryologia 1(52) s209-222. 9 1949. Early Influence oi' m-inositol and Sugars oi' Gammexane Induced c-mito-sla* CaryoLagia 1(2):223-228. ______________________________ . ____________ y 1950. Notes on the Chromosome breaks induced by Pure Gammexane. Caryologia 2.;561-564. Daneil, A. and G. B* Wilson, 1954. (in press) The Antimitotic Eii'ect of Endothal. Frear, D. E. H., 1949. Chemistry ol‘ Insecticides. Fungicides and Herbi­ cides. D. Van Nostrand Co., Inc., N. I., 1949. Gaulden, M. E. and J. G. Carlson 1951. Eii'ects of Colchicine on the Grasshopper Nearoblast. Expmt. Cell Res* 2:416-435. Greenwood, M. L. and J. M. Tice. Palatobility Tests on Potatoes Grown in Soil Treated with the Insecticides Benzene Hexachloride. Chlordane and Chlorinated Comphene. Jour. Ag. Res. 78(11)t477-482. Guttman, R., 1952. An Interpretation oi‘ Some Mitotic Irregularities Using Poison Distribution. Amer. J. of Bot. 39(8);528-534. Havas, L. J., 1949. Hormone-mimetic and Growth Eilects ox Colchicine. Exp. Cell Res. Suppl. 1:597-601. Hawthorne, M. E., and G. B. Wilson, 1952* The Cytological Effects of the Antibiotic Acti-dione. Cytologia 17*71-85. Himdraarsh, M. H., 1952. The Effect of Colchicine on the Spindle of Root Tip Cell. The Proc. of Linn. Soc. 77;500-506. Hober, R., 1945. Physical Chemistry of Cells and Tissues* Co. Philadelphia, 1945. The Blakiston Hocking, B., 1950. On the Effect of Crude Benzene Hexachloride on Cereal Seedlings. Bci. Ag. 50:165. Huskins, C. L. and L. N. Steinitz, 1948. The Nucleus in Dilierentiation And Development. _Jour. Hered. 39;66-77. Huston, M. J., 1952* Cytological Effects of Certain Organic Chemicals. Unpublished M.S. Thesis. Michigan State College, 1952. 60 Kirkwoua, S. and p. H. Phillips, 1946* Tne Aatiinositol Effect ol j ^ exachlorocycf ohexane. J- Biol* Chem. 165:261-254. Kostoff, D*, 1948* Cytogenetic Changes ana Atypical Growth Induced by Ben&ene Hexachloride. Curr. Bci. 10:17. ____________ , 1949. Atypical Growth. Abnormal Mitosis* Polyploidy and Chromosomal Fragmentation Inaneea by Hexachlorocyclohexane. Nature 162:845-846. ____________ , 1949. Inauction of Cytogenetic Cimn^es and Atypical Growth by Hexachlorocyclohexane. Science 10 9 ;467-468. Levan A., 1958. The Effect ol Colchicine on Root Mitoses in Allium. Hereditas 24:471-486. m _________, and G. Ostergren, 1943. The Mechanism ol c-mitotic Action Observation on Naphthalene Series. Hereditas 29;531. _________ , and H. J . Tjio, 1948. Induction oi Chromosoiae Fragmentation by Phenols. Hereditas 54:455-484. _________, and T. Lotfy, 1950. Spontaneous Chromosome Fragmentation in Seedlings of Vicia Faba. Hereditas 56:471-482. ________ , A., 1952. Chemically Induced Chromosome B.eactions in Allium Cepa and Vicia F a b a . Cold Spring Harbor Symposium. Quant. Biol. 16:255-242. ________ , 1954. Colchicine Inauced c-mitosis in Two Mouse Ascites Tumours. Hereditas 40:1-64. Macfarlane, E.W.E. and N. G. Schmock, 1948. The Colcnicine and ColchicineLike Reaction as a Possible Response to Enzymic Poisoning. Science 108:712-713. Nybom, N • ana B. Knutsson, 1947. Cepa. Hereditas 35:220-254. Investigation on c-mitosis in Allium Ostergren, G. and A. Levan, 1946. The Connection Between c-mitotic Activity and. Water Solubility in Some Monocyclic Compounds. Hereditas 2 9 :381-443. 1950. Cytological Standards for the Quantitative Estimation of Spin dle Disturbances.. Hereditas 36;571— 582. , 1950. r.nmilderation on Some Elementary Features of Mitosis. Hereditas 56:1-18. 61 Ostergren, G. ana A. Levan, 1961, Narcotized M i tosis and the Precipita­ tion Hyp o thesis 01 Narcosis. Colloqu.es internet, do la centre national de la recherche scientiiique. 26:77-87. Powell, S. S., 1951. Comparative ElTects ol Colchicine ana Various Nucleic Acid Salts upon Somatic Mitosis • Unpub iii heu M.S. thee i n . Michigan State College, 1951. Rao, N. S. and B. C. Kundu, 1949. EiJtect of Gammexane on the Root Tips oi Corchorus C&psularis L » Science and culture 14(11) *484. Scholes, M.E., 1963. The Eflect ol Hexachlorocyclohexane on Mitosis in Roots of Onion and Strawberry (Fragaria V esca). Jour. Hort. Sci. 28(1):49-68. Schopfer, W. H», Posternak, T. and M.L.Boss, 1947. Le Gammexane ( y-hexachlorochohexane) est-il 1 1antivltamine du mesoinositol? Schweiz. Zeitschr. f. Pathol, und Bakteriol. 10:443-417. Shepard, H. H., 1951. The Chemistry and Action oi Insecticides. Hill Book Co., Inc. 1961. McGraw- Sigenaga, M . , 1949. Experimental Studies oi' Abnormal Nuclear Cell Divisions. Cytologia 16:45-60. Slaae, R. E. 1945. A new British Insecticide, the Gamma Isomer ol Benzene Hexochlorid. Chem. and Ind.. 64:514. Stitt, L. I. and J. Evunson, 1949. Phvtotoxicity and Qi'i'-Quality of Vegetables Grown in Soil Treated with Insecticides. Jour. Econ. Ent. 425:615-617. Stoker, R. I. 1948. The Phytotoxicity of DDT and Benzene Hexachloride. Ann. Appl. Biol. 55;110-125. Vaarama, A., 1947. Experimental Studies on the IniMuence of DDT Insecticide Upon Plant Mitosis. Hereditas 55:191-219. Von Rosen, G., 1953. Radiomime-tic Activity and the Periodical System oi’ the Elements. Bot. Notiser 1955 (1):140-142. West, T. F. and G. A. Campbell, 1950. PUT and Newer Persistent Insecti­ cides. Chapman and Hall Co. London, 1950. Wilson, G. B., M.E. Hawthorne, ana T. M. Tsou, 1951. Spontaneous and Induced Variations in Mitosis* Jour. Hered. 42*183-189. 62 ' Yiil&ou, G. B., T. M. Tsou ana P. Hvypio, 1952. Variations in Mitotis. II. the Interrelation of Some Basic Deviations . Jo;ir. Hered. 4'6:211 -2 15 • Woolley, D. W., 1952. A Stuuy of Aatimetabolites. Inc., Nev; York, 1952. John Wiley and ion PIATE I Control and -Affected' Mitotic Stages ol Allium Cepa Figures: T,, Bpi'eaa segregated propnase with two trugiiients cum two diceutxic chromosomes taken alter triree hours treatment with 1,000 ppm sus­ pension. 2. Segregated c-metaphnse taken alter nine hour treatment with 10 ppm suspension. 3. Segregated apolar anaphase taken after four hours, treatment with 1,000 ppm suspension. 4. Over-contracted prophase suspension. 5. Spread metaphase taken after 30 minutes treatment w ith 1,000 ppm sus­ pension. 6. Scattered metaphase taken after six hours treatment with 500 ppm sus­ pension. 7. Apolar anaphase taken after five hours treatment with 8. Normal tetraploid metaphase taken after 48 hours recovery treatment with 150 ppm suspension. 9. Normal polyploid anaphase and resting stage after 48 hours recovery of 24 hours treatment with 150 ppm suspension. taken after 24 hours; treatment with 1,000 ppm 500 ppm suspension. of 24 hours 10. Clumped metaphase with two fragments taken after 5 ))eHrs treatment with 1,000 ppm suspension* 11. Normal anaphase with two fragments taken from untreated material. 12. Micro-nucleus taken from untreated material. 13. Normal prophase taken from untreated material. 14. Normal metaphase taken from untreated material. 15. Normal early anaphase taken from untreated material. 16. Normal anaphase and telophase taken from untreated material. Each division of the scale represents ten microns. * i p 1 A* if I I & W I PLATE I £ PLa TE II Affected Mitotic Stages of Allium Cepa Treated With 1.000 ppm Su p punsion Figures: 3. Spread segregated prophase tahen after 30 mi mites treatment. 2. Segregated contracted metaphase taKen after 30 minutes treatment. 3. Tetrapolar anaphase tamen after 15 minutes treatment* 4. Early "star1* bipolar anaphase taken after 30 minutes treatment. 5. "Star'' bipolar anaphase taken after 15 minutes treatment. 6. "Star" bipolar anaphase taken after 30 minutes treatment. 7. Early multipolar anaphase taxen after 15 minutes treatment. 8. Multipolar anaphase taken after 3 hours treatment. 9. Trinucleate cell tanen after 24 hours recovery of 3 hours treatment. Each division of the scale represents ten microns. 64 m» z~ ^ I ^ 3 '■ _ ft. £ > 2 v V , cjr' j * # * f t ^ * . # 1 4 r * # ^ *k n 5 1 j . < 1 6 « v• * > ■ , » PLATE II ■ * ■ i ■ ■ j I FLa TE i/jiteres ox binnclsate Cell a o III 1__allimn Cepa TaKen I rom 47 Hoars Recovered Root-Tips Alter 1 Hour Treatment Wi_th_ 1,000 ppm fiispension Figures: 1. Prophase 2. Prometaphase 5. Early anaphase 4. Anaphase C Telophase Each divirion of the sca_le represents ten microns. 65 r \ PLATE III PLa TE IV Mitotic Stages From Untreated Pi sum Sativum Figures: 1. Interphases. 2. Early prophases. 5. Midprophase. 4. Late prophase. 5. Late prophase, prometaphase, and early prophase. 6-7* Early prometaphase. 8. Prometaphase. 8. Prometaphase with a lagging chromosome. 10. Prometaphase and early anaphase. 11. Early metaphase and prometaphase. 12. Early ana late prophase, and ea.rly metaphase. 13-14. Metaphase. 15. Early prophases, and anaphase. 16. Telophase with two lagging chromatids. Each uivision ol the scale represents ten microns. 66 J0L I •#1 2 KJ/. *.> * PLATE IV *'■ PLnTE V Alloc-,tea Mitotic glares in Pi^um gativum Treated with 1,000 ppm Lux,pen Lion Figurel : 1. Clumped metuphase and normal prometaphase taken alter one hour treat­ ment. 2. Spread and segregated prophase, clumpea telophase taken alter four hours treatment. 3* Scattered metaphnse taken alter lour hours treatment. 4. Apolar anaphase taken alter one hour treatment. 5. Biriucleate scattered metaphase taken alter £4 hours treatment. 6. Binucleate prophase with one Iragmental nucleus taken alter 24 hours treatment. 7. Apola.r segregated polyploid anaphase taicen alter 24 hours treatment. 8. Nine nucleate cell taken alter 24 hours treatment. 9. Polyploid apola.r anaphase taken alter 24 hours treatment. 10. Tetraploid scattered metaphase taken alter 24 hours treatment. 11. Octoploia scattered metaphase taken alter 24 hours treatment. 12. Tetraploid normal prophage ana metaphase taken alter six days re­ covery alter eight hours treatment. Each aivision ol the scale represents ten microns. 67 >> vf; J&P n < ym « & m ♦ i * 3 r * i t i *> I 8 «f f I % «. r* I 4*> i?^2£ 10 % w 1 Normal r—1 U Spread 3 Segregated a Disorganized Spread +* Scattered <11 a Segregated Clamped Normal.. Normal ?! Star bipolar a. Multipolar o fH Apolar a> E-t Apolar+seg* M:Ltotic index Bl.multinucleate CEPA 15 G) O 1Normal 31 S a 21 Spread 1 o u Segregated — cu |Overcontracted— to 4 1 — — & Clumped ProphS.se Metaphase Anaphase Telophase l — 6 l 8 12 — 5 l 8 9 — l 7 8 l 10 l 12 9 10 11 14 i i i l i 2 9 8 19 11 1 _ — 5 3 3 3 2 9 — — — — 1 — -- — — — — — — — — 24 — — 3 __ __ 4 — — — 1 5 6 3 3 — — _______ 6 — __ _____ ____ — —— —— __ —— 30 29 38 27 27 25 28 12 16 16 8 10 — — 2 2 2 — 1 — 3 — 1 — 2 2 7 7 10 14 17 12 31 14 17 26 29 50 1 -- — — — — — 2 2 1 2 5 7 -3 — — 1 — 3 — — 11 57 13 9 21 — — 6 5 — 4 — 2 3 — — • — 2 10 2 1 — — — 12 2 — — — — 10 — — — 12 — 1 — 4 — 9 1 — — — 9 12 — 2 — 1 — 8 9 — 2 1 — — — — — — 54 44 36 36 33 35 38 47 37 43 4 11 8 15 14 7 7 9 13 10 _ __ — 1 — 9 Mitotic index 4 . 4 Bi, multinucleate — l .— — a o« Apolar 1 1 —oI Apolar+seg. — © E-» 4 1 Normal 3 Disorganized 3 Polar — A-polar 5 A-polar+seg.— Clumped — Normal 4 27 25 24 22 21 21 20 12 13 25 25 15 ____ ________ ________ ____ i — 1 l l— — 1 — — — — 1 -- — — — — ____ ____ ____ ____ ____ ________ Normal — Disorganized 3 Spread 10 Sp.+seg. — -§p• Scattered 0 OC#TS0g# — Clumped — Ball a c> a % 3« 5 15 9 + -4 -4— 1 — — — — - 9 — 7 — — — — — — — — — 10 3 — _ 14 18 — — — — — - — — 14 13 3 2 8 — ' 4 .9 _ — 1 — 33 41 15 £3 38 42 40 46 31 37 14 9 .15 21 10 11 10 24 25. 15 5,5 7,5 6,5 6.9 5.6 6.1 7.01,3 — v —- — ■ — — 21 — — — — 2 1 — 3.5 4 — — 48 34 14 4 4 .5 — 7 10 3 5 — — 30 40 18 12 17 57 14 12 3.3 4 .5 4— 4----- =fe=L----- £----- ±----- 4----- 4— ±444 71 APPENDIX Ta b l e IV PERCENTAGE DISTRIBUTION 01' MITOTIC STAGES IN RECOVER! RUN .01 a L L U M £ERa Time (hr.) 1 1 No.of elide 1 1 Krf Normal Spread Segregated 25 39 5 Segregated — Overcontract — Ja-H c]xt Spread o Normal Spread 5 Segregated 2 1 Normal 2 a> Disorganized — 03 A $> + 0) Spread Scattered Segregated Clumped Normal Disorganized & Multipolar §• Apolar Apolar+seg. A 1 27 1 — 1 6-» normal Multipolar Apolar Apolar+seg. Prophase Metaphase -P o Anaphase e-i Telophase 1 1 1 1 22 17 22 23 26 ly 15 16 13 21 12 23 — — — — — — — — — — — — — — — — — — — — 3 —■ 23 5 — — 15 3 — — 16 1 — — 22 — — — 27 — 1 — 20 — 1 — 31 — — — 32 — 1 — 54 — — — 32 — — — 28 — 2 — 26 — — — 10 — — — 21 — — 2 — 1 — 2 — — 1 — 5 — — — — 29 — — 24 13 — — 10 11 — — — — — 1 2 — 5 14 3 — — ■ 9 12 1 V CO $ Oi o r-\ to 1 — — 1 — 3 2 1 — — 11 — 6 1 1 1 — — — to to 1 14 -• — — — formal 7J Spread Segregated I 27 0) 1Normal T3 Spread 1 i .3 a ■a Segregated o f-t 0ver-contrac ta, Normal Vil +3Spread aJ Segregated Normal Al Disorganized Q CQ Spread 3O- Sp.+ seg. 3 Scattered © Sc.•»* seg. 33 Clumped Ball © 0) 1 2 . — — . 5 ---- • • 4 8 24 -.. --- --- — 2 2 2 2 2 2 2 29 — - 29 — - 26 25 25 40 28 - - - - - 16 — — —* 24 26 24 24 20 20 5 — n . _ _ _ 5 3 7 1 6 1 - - - 19 1 - 20 1 2 17 — 16 — — 1 1 — — - - - — - —- — — — - 6 1 9 — __ - 20 — — — — 5 — — - — — 1 1 — — 1 — 19 1 7 - 3 10 _ 1 — 8 5 — - - Normal 9 Disorganized 1 Polar Apolar Apolar4seg. - 6 — - - 19 17 1 — — — — — - — - 8 1 1 _ _ — — — - 6 8 - — — — - — _ - - — — — — 1 17 13 16 15 11 - — — — —■ — — 14 — • — — — - - - — — — — - - —■ — Prophase Metaphase o Anaphase IH Telophase 62 20 10 61 23 62 18 59 17 64 63 59 18 7 8 9 17 Mitotic index 6.3 Bi- .multinucleate - Normal i« O 03Polar fH Apolar © JS tr-ia Apolar4seg. rH -3 4 0 +3 8 _ 9 13 8 16 21 6 9 55 21 9 15 20 6 11 6.2 6.5 5.1 5.5 5.0 7.4 4.1 - - - - - — 9 14 73 appendix table vi PERCENTAGE DISTRIblJTIuN up MITuTIC STAGES' IN CuNTlNUuUS TREATMENT OF PI SUM SATIVUM. 2 Time(hr.) 0 co 8 CL, No. of slide 4 4 Normal Spread Segregated 20 22 Normal 35 Spread 6 Segregated — Overcontracted— S' 3 0 V J N o rmal Disorganized Spread Sp.+seg. Scattered Sc.+seg Clumped Ball t ■3 o EH Prophase Metaphase Anaphase Telophase Mi totic index Bi multinucleate 8 24 24 13 4 16 28 9 18 It 3 6 2 1 4 8 1 27 2 — 1 1 11 1 10 3 1 12 3 — 1 — 2 — 6 2 — — 2 1 1 1 8 1 66 15 10 9 7.0 ±__ 23 18 1 1 3 3 3 4 1 — 1 10 2 10 — — — 9 11 — 7 1 1 3 1 6 3 6 3 — 2 — 24 9 5 — — — — — — — — — — — 4 3 4 2 2 4 45 39 9 7 53 31 42 40 10 6 12 6; 5.5 5.5 6.8 10.6 *+ ++t ++++ ^ ++-*-+ 2 2 62 23 9 51 30 50 31 10 10 9 9 6.1 + . — 1 6 3 _±_ — — 1 12 9 7 1 8 1 7 6.3 - — 2 6 . — ' 10 — 3 1 — 1 5 3 3 3 — — — — — 3 — 1 2 1 1 2 5 — 21 6 — — — 2 — 26 3 i l 1 Normal Disorganized Polar Apolar Apolar+seg. 03 - Normal Polar Apolar 1 Apolar+seg. 4 3 Normal Spread Segregated CO 3