Rapid Action of Carbamate Herbicides Upon Plant Cell Nuclei

Transcription

1 Rapid Action of Carbamate Herbicides Upon Plant Cell Nuclei Jay D. Mann1 and William B. Storey Department of Horticultural Science, University of California, Riverside, California 92502, U. S. A. Received October 5, 1965 Carbamate compounds, two of which are IPC (O-isopropyl N-phenyl carbamate) and CIPC (isopropyl N-(3-chlorophenyl) carbamate) have been in use since the late 1940's as selective herbicides2. They are known to be extremely toxic to newly germinated seedlings of monocotyledonous plants. They are toxic to certain dicotyledonous plants, also, but to a lesser degree. They tend to stunt plants by inhibiting their growth, rather than by killing them outright. Early reports indicated that the nuclei of affected cells were seriously disorganized after 1 to 7 days of treatment with IPC or CIPC concentrations of 1 to 100 ppm in aqueous solutions. Shorter periods of treatment were not tested (Ennis 1948, 1949, Scott and Struckmyer 1955). Recently, we reported that CIPC inhibited the incorporation of leucine-1-c14 into protein by excised seedling tissues within one hour after treatment (Mann, Jordan, and Day 1965). Certain features of nuclear abnormalities caused by IPC and CIPC re semble those produced by colchicine, leading to the description in the literature of these herbicide-treated cells as "c-mitotic." Consequently, one of us (W. B. S.) decided to see if the carbamates might have some use in cytological studies. Two facts immediately became apparent. First, the response of meristematic root-tip cells to low concentrations begins within the first half hour of treatment, leading rapidly to complete cessation of all mitotic activity. Second, the carbamate herbicides contract chromosomes, but the configura tions do not resemble those produced by colchicine. The more-or-less cruci form configurations typical of c-mitosis are not formed. We followed our initial observations with a series of experiments with IPC upon root tips of the broad bean (Vicia faba), onion (Allium cepa), calla lily (Zantedeschia aethiopeia), Macadamia nut (Macadamia tetraphylla), and papaya (Carica papaya). Concentrations of 2.5, 5, 10, 20, 40, and 80 ppm were used, and root tip samples were withdrawn after treatments for 0.5, 1, 2, 4, and 8 hours. The root tips were killed immediately in a solution 1 Present address: Monsanto Company, St. Louis, Missouri, U. S. A. 2 Purified IPC and CIPC are available from F and M Scientific Corporation, Avondale, Pennsylvania.

2 204 J. D. Mann and W. B. Storey Cytologia 31 of 3 parts of 95% ethanol and 1 part of glacial acetic acid. Nuclei were stained by the widely used method of squashing the root tips in aceto-orcein. Chromosomes at the metaphase stage in treated root tips were compared visually with their counterparts in untreated root tips to ascertain the approximate degree of contraction as a function of concentration and time. Contraction and final disintegration of the chromosomes of Vicia faba are shown in Fig. 1. This species was chosen as the example for illustra tion simply because it has fewer (2n=12), larger, and more easily recognized chromosomes than most other plants. Fig. 1, A, shows the chromosomes in a root tip killed in acetic acid-alcohol without pretreatment. Fig. 1, B, shows the chromosomes in a root tip from the same plant after 1 hour in 10 ppm IPC. Fig. 1, C, shows the chromosomes in a third root tip from the same plant after 2 hours in 10 ppm IPC. Fig. 1, D, shows the chromosomes after 4 hours in 10 ppm IPC. Fig. 1, E, shows late anaphase chromosomes clumped together and beginning to disintegrate after 5 hours. The divided chromo somes are becoming granular and will form into nuclei which are indis tinguishable from those that had been in the metabolic state. All other states of mitosis share the same fate (Fig. 1, F). The chromosomes in Fig. 1, B, are reduced to about 75 per cent of the lengths of their counterparts in Fig. 1, A, those in Fig. 1, C, to about 65 per cent, and those in Fig. 1, D to 50 per cent, or less. The action of IPC is not strictly comparable with that of colchicine, for its effect upon the chromosome appears to be tightening of the spiraling chromonemata accompanied by increased density and darker staining of the matrix, as well as cessation of all mitotic activity. Character istic c-mitotic cruciform chromosomes, with well separated arms but firm attachment at the centromere are not found. There is little or no con traction of the divided chromosome in true c-mitosis. The chromosomes in Fig. 1, D are at the point where destruction of the chromatin is beginning. High concentrations of IPC, and longer periods of treatment even at very low concentrations, lead to disintegration of the nucleus. Mitotic metaphase chromosomes tend to meld into an amorphous mass followed by granulation and disintegration. Metabolic nuclei stain more intensively, with the heteropyknotic regions of the attenuated chromo somes becoming granular in appearance. The effects of IPC described above are also induced by CIPC and by barban (4-chloro-2-butynyl N-(3-chlorohenyl) carbamate). We do not know yet if withdrawal of the herbicide treatment will be followed by resumption of normal mitotic processes or if restitution nuclei will result. In a similar series of experiments that came to our attention after the completion of this work, Doxey (1949) kept roots of onion and rye in IPC solutions before transferring to distilled water. Under these conditions, a pretreatment of onion roots with 50 ppm of IPC caused "over

3 1966 Rapid Action of Carbamate Herbicides Upon Plant Cell Nuclei 205 contraction" of chromosomes, but the nuclei of rye were still quite dis organized. In contrast, our findings indicate that overcontraction occurs very soon after IPC treatment, and that it occurs for every species tested. Despite the apparently tetraploid appearance of some nuclei after carbamate treatment, we have been unable to the present to induce polyploidy in a number of plant species that have responded previously to colchicine. The carbamate herbicides hold promise as a new group of chemicals for use in cytological researches, especially determinations of chromosome numbers and morphology. Cytologists often find these tasks arduous, and sometimes impossible, in species having long, stringy and numerous chromosomes having Fig. 1. Effects of 10 ppm IPC in aqueous solution on Vicia Jaba chromosomes following treatments for increasing lengths of time. Magnification ca. ~1665, except F, which is ~ 665. A, no treatment. B, 1 hour, ca. 25% contraction. C, 2 hours, ca. 33% contraction. D, 3 hours, ca. 50% contraction; onset of disintegration. E, 4 hours; clumping and pro gressive disintegration of late anaphase. F, 5 hours; complete disintegration and granu lation; cells in all stages of mitosis indistinguishable from cells in metabolic stage. arms that cross which tend to resist spreading in squash preparations. This sort of problem accounts for many of the disparities in chromosomes numbers reported by different workers for the same species (Darlington 1956, Cave ). In order to achieve chromosome separation (in some cases ac companied by contraction), many cytologists resort to the use of certain chemical compounds, principally colchicine, 8-hydroxyquinoline, and para dichlorobenzene for treating root tips prior to killing and fixing for staining. We found none of these, as well as several others commonly in use, to have any appreciable effect on chromosomes of numerous species of Araceae. Cytologia 31,

4 206 J. D. Mann and W. B. Storey Cytologia 31 Jones (1963) and Freytag (1964) reported that they fail to serve the purpose in the Orchidaceae. The carbamates, on the contrary, have caused contraction of chromosomes, and usually good separation, in every species upon which we have tested them. IPC and CIPC might well supersede colchicine for use in cytological studies for several reasons. First, they achieve the effect for which colchicine is primarily used; i.e., improved visibility of chromosomes. Second, in the light of present knowledge, there is good probability that they will affect a wider range of species. Third, their mammalian toxicity is extremely low (for IPC, oral LD50 is 3000mg/kg body weight). Fourth, colchicine is classed as a carcinogenic substance, whereas IPC produced neither benign nor ma lignant tumors in adult rats and mice to which it was administered in clinical experiments during an 18-month period. Fourth, IPC and CIPC are much more economical to use than colchicine. All three substances are priced at about $ 3.00 a gram in pure form. However, our studies suggest that 10 ppm of a carbamate produces results comparable to 1000 ppm of colchicine, implying a 100-fold difference in cost when used. Our studies, as well as those reported by Ennis, Scott, and Struckmyer, suggest that these compounds deserve both more comprehensive and more in tensive investigation for ascertaining their real potential as tools in cytological researches, not only on plants but also on animals, especially in the field of nucleus-cytoplasm interactions. The rapid contraction of metaphase chromo somes, together with the graininess of the chromatic regions of the attenuated chromosomes in metabolic nuclei, suggest that the DNA of carbamate-affected chromosomes may be unable to function as templates for the synthesis of messenger RNA. Entirely unknown at this writing, of course, is what effect, if any, do those herbicides have upon ribosomes, mitochondria, cytoplasm, and other extra-nuclear components of the cell. It is interesting to note one commercial use of CIPC. Tuan and Bonner (1964) demonstrated recently that sprouting of potatoes was accompanied by nuclear activation. Potato tubers sprayed with CIPC will not sprout, although such treated tubers retain metabolic activity, as indicated by continuing re sistance to fungal infection. Abstract IPC (O-isopropyl N-phenyl carbamate), CIPC (isopropyl N-(3-chloro phenyl) carbamate), and barban (4-chloro-2-butynyl N-(3-chlorophenyl) carba mate) are selective herbicides which inhibit growth of young seedling plants without causing immediate death. We find these compounds useful in making determinations of plant chromosome number and studies of their morphology in root tips. Treatment is with aqueous solutions ranging from 2.5 to 80 ppm for periods of 0.5 to 6 hours, followed by staining by the aceto-orcein squash method. IPC and CIPC cause mitotic activity to cease almost immediately.

5 1966 Rapid Action of Carbamate Herbicides Upon Plant Cell Nuclei 207 Chromosomes undergoing mitosis contract, in all species of plants studied, including some that have been reported to show no response to colchicine, 8-hydroxyquinoline, pares-dichlorobenzene, and other chemicals used for this purpose. Metabolic nuclei show changes, also, suggesting that the herbicidal action of these compounds may be due to inhibition of messenger RNA synthe sis by the DNA template. Uniformly good results to date on many species of plants suggest that IPC and CIPC may well replace chemicals now com monly used for facilitating cytological studies of plant cells. They are de cidedly less hazardous to health than colchicine, and considerably cheaper to use. Acknowledgment The work with CIPC and barban was performed by Mr. Edward Selinger during an NSF Summer training programs. This research was supported in part by NIH Grant GM , and by a donation from the Pittsburgh Plate Glass Company. Bibliography Cave, M. S., Ed Index to chromosome numbers of plants. Vol. 102, Univ. of No. Carolina Press, Chapel Hill. Darlington, C. D. and Wylie, A. D Chromosome atlas of flowering plants. Macmil lan, New York. Doxey, D The effect of isopropylphenyl carbamate on mitosis in rye (Secale cereale) and onion (Allium cepa). Ann. Bot. 13: Ennis, W. B., Jr Some cytological effects of O-isopropyl N-phenylcarbamate upon Avena.A. J. Bot. 35: Histological and cytological responses of certain plants to some aryl carbamate esters. A. J. Bot. 36: 823. Freytag, A. H The use of ethanol to improve aceto-carmine staining of orchid chromosomes. Stain Tech. 38: Jones, K The chromosomes of Dendrobium. Amer Orchid Soc. Bul. 32: Mann, J. D., L. S. Jordan, and Day, B. E The effects of carbamate herbicides on polymer synthesis. Weeds 13: Scott, Marie A. and Esther Struckmyer, B Morphology and root anatomy of sqnash and cucumber seedlings treated with isopropyl N-(3-chlorophenyl) carbamate (CIPC). Bot. Gaz. 117: Tuan, D. Y. H. and Bonner, J Dormancy associated with repression of genetic activity. Plant Phys. 39: *