INSTITUTO BOTÂNICO DA UNIVERSIDADE DE COIMBRA BOLETIM DA SOCIEDADE BROTERIANA ( FUNDADO EM 1880 PELO DR. JÚLIO HENRIQUES) VOL XLVII (2.A SÉRIE) REDACTORES PROF. DR. A. FERNANDES Director do Instituto Botânico DR. J. BARROS NEVES Professor catedrático de Botânica COIMBRA 1973 BOLETIM DA SOCIEDADE BROTERIANA VOL. XLVII (2.ª SÉRIE) 1973 INSTITUTO BOTÂNICO DA UNIVERSIDADE DE COIMBRA BOLETIM DA SOCIEDADE BROTERIANA (FUNDADO EM 1880 PELO DR. JÚLIO HENRIQUES) VOL XLVII (2.A SÉRIE) REDACTORES PROF. DR. A. FERNANDES Director do Instituto Botânico DR. J. BARROS NEVES Professor catedrático de Botânica COIMBRA 1973 omposição e impressão das Oficinas da c Tipografia Alcobacense, Lda. — Alcobaça THE EFFECT OF N20 ON MEIOSIS by J. MONTEZUMA-DE-CARVALHO * Botanical Institute, University of Coimbra INTRODUCTION NITROUS oxide (laughing gas, N20) has been found to induce c-mitosis in root tips of Pisum sativum at atmos- pheric pressure (ÕSTERGREN, 1944) and in Allium cepa at a pressure of six atmospheres (PERGUSON et al., 1950). Based on this c-effect N20 has been applied, by several authors, to zygotes at the time of its first cleavage in order to obtain polyploid plants (ÕSTERGREN, 1955 in Crepis capilla- ris; NYGREN, 1955 in Melandrium; ÕSTERGREN, 1957 in Pha- laris; KIHARA and TSUNEWAKI, 1960 in Triticum; ZEILING and SCHOUTEN, 1966 in Tulipa). It has also been shown that N20 readily induces, under pressure, c-mitosis in the pollen tubes in styles (MONTEZUMA-DE-CARVALHO, 1967). As far as we know no Work has been done on the effects of N20 on meiosis. In the present paper we describe some results that demonstrate that this gas can drastically change the course of meiosis by its property of spindle inhibition (c-effect). MATERIAL AND METHODS Anthers of Tradescantia paludosa (2n = 12) were used in our experiments. In this plant as in Tradescantia bracteata (MONTEZUMA-DE-CARVALHO, 1957) meiosis is fairly well synchronized in the pollen mother cells of an anther and * Trabalho subsidiado pelo Projecto de Investigação Científica CB1 do Instituto de Alta Cultura. [5] 6 J. Montezuma-de-Carvalho it is possible to dissect the two lobes of it. Accordingly one lobe (half-anther) was treated with N20 and the other was used as a control to indicate the stage of meiosis at the begining of the treatment. Anthers at the stage of prome- taphase I, metaphase I and metaphase-anaphase I were choosen for treatment. The lobes, were put in open Petri dishes on a piece of wet filter paper. The gas apparatus used was of the same type as recommended by ÕSTERGREN (1955) and the N20 treatments were applied according to the following schedules: 5 atmospheres/4 hours; 9 atmos­ pheres/4 hours; 6 atmospheres/22 hours. For each expe- riment four lobes were treated. Pollen mother cells were stained with acetic-orcein and mounted in Euparal, each slide containing the cells of a single lobe. RESULTS AND DISCUSSION From the analysis of Table I and Plates I, II and III we can conclude the following facts: a) cells at the stage of prometaphase I treated with N20 at 9 or 5 atmospheres during 4 hours show at the end of treatment either the six bivalents in a diakinesis-like appearance (Plate I, fig. 1) due to the abortion of the metaphase plate or the homo- logous chromosomes of a bivalent in a c-anaphase I (Plate I, fig. 2 shows just the beginning of the c-anaphase separation and fig. 3 and 4 the chromosomes already fully separated). In these figures it is clear that in the absence of a spindle the two chromosomes of each bivalent remain in the vicinity of each other, demonstrating that, whatever the forces of a normal anaphase-I movement may be, one component of such a movement must be an initial repulsion between the chromosomes themselves as postulated by DARLINGTON (1937). It is evident also, from these figures, that the cen- tromere is not divided contrary the opinion of LIMA-DE-FARIA (1956) who admits that it is already divided at anaphase-I of meiosis. A strong sister-chromatid repulsion is also apparent at this stage; b) cells treated at metaphase I during 4 hours at 9 atmospheres are seen at restitution The effect of N20 on meiosis 1 interphase (c-interphase). A complete analysis of one slide (correspondent to an half-anther) with ca 450 cells showed that most of the cells (90 %) were in a complete restitution, i. e., all the chromosomes are included in one restitution nucleus. These nuclei are of three types: rounded or lobu- TABLE I Effect of N20 on meiosis in Tradescantia paludosa Pressure and Time for Stage after treatment Stage treated duration of recovery or recovery treatment Prometaphase-I 5 atmospheres zero Diakinesis-like /4 hours bivalents; c-anaphase I Prometaphase I zero Diakinesis-like bivalents; c-anaphase I Metaphase-I 9 atmospheres zero restitution nuclei /4 hours (c-interphase) Metaphase-Ana- 16 hours Diploid prophase- phase I -prometaphase II; normal tetrads Prometaphase-I 6 atmospheres zero Arrested c-meta- /22 hours phase-anaphase I; Intense stickyness lated nuclei (Plate II, fig. 1), ring-shaped nuclei (Plate II, fig. 2) and sausage-shaped nuclei (Plate II, fig. 3) appearing with a f requency of 71 %, 17 % and 2 % respectively. The comparatively high frequency of ring-shaped nuclei results from the more or less circular disposition of the bivalents (Plate I, fig. 1) with the consequent disposition of the chromosomes at c-anaphase-I in a ring (Plate I, fig. 3), and if the ring is not closed at c-interphase it will give rise to a sausage-like nucleus (Plate II, fig. 3). An example of an incomplete restitution nucleus is seen in Plate II, fig. 4 where a chromosome lies in the cytoplasm. This loss of a 8 J. Montezuma-de-Carvalho chromosome appears in 9 % of the cells. A very few cells (I %) were seen with two non included chromosomes, and cells with three chromosomes were not seen; c) cells treated at metaphase-anaphase I during 4 hours with a pressure of 9 atmospheres and then allow to pursue the meiotic cycle during 16 hours were found to be in a diploid prophase- prometaphase II (Plate III, fig. 1, 2 and 3). Obviously, these cells derive from the c-interphase cells produced during the N20 treatment. Unfortunately we have not observed those cells at anaphase II due simply to the fact that 16 hours recovery at ± 19° C was not enough to allow the cells to reach this stage. It is known (MONTEZUMA-DE- -CARVALHO, 1960) that in normal half-anthers of Traães- cantia paludosa put at 25° C the interval between meta- phase I and telophase II is of the order of only 7 hours, so, a lower temperature combined, possibly, with the effects of N20 hás the effect of increasing this time interval. But a proof that a functional spindle can be again organised, after the treatment, is the fact that in the same slides a few cells were seen in normal tetrad stage. These cells are the derivatives of the ones that were already at ana­ phase I at the time of the treatment and therefore its spindle had no time to be affected by the gas. We can conclude then that if cells are treated at prometaphase- -metaphase I and then allow to recover at the right tempe- rature-time interval, diploid pollen dyads will be obtained. Diploid dyads will also be formed if the gas treatment is made at the time of the normal prometaphase-metaphase II. Tetraploid pollen monads would be produced if N,0 treatment is applied during enough time to affect both the first and the second division, but in our material this was not observed because of the toxic effects of N20. In fact (Table I) cells treated at prometaphase I with 6 atmos­ pheres during 22 hours, were arrested at c-metaphase- -anaphase I, due to cell death. This lethal effect is noti- ceable by the intense chromatic agluttination and picno- citicy of the bivalents (Plate III, fig. 4). Judging from the viability of the cells that were treated at the same stage during only 4 hours we can conclude that a treatment of The effect of N20 on meiosis 9 4 hours is at the limiar of time-dosis permissible to the meiotic cells of Tradescantia paludosa. The lethal effect could, probably, be overcome by a fractionated treatment, i. e., N20 applied twice with an intermediate stage (c-inter- phase) for recovering. This seems rather possible as it was found (MONTEZUMA-DE-CARVALHO, 1967) that a treatment of N20 during 2 hours only, is enough to produce spindle inac- tivation on pollen tubes growing in styles. So, a 4 hour treatment fractionated in two, each of 2 hours, would not be lethal and would produce tetraploid pollen monads, if applied at the right stage (firstly at prometaphase-meta- phase I and secondly at diploid prometaphase-metaphase II). It is interesting to compare the effects of N20 and of cclchicine on meiosis. The action of colchicine on meiosis has been studied by WALKER (1938), SHIMAMURA (1939), BARBER (1942), PRAAKEN & LEVAN (1946), MONTEZUMA- -DE-CARVALHO (1962). As both agents, N20 and colchicine, are spindle inhibitors, it is expected that the main action on meiosis will be the same, i. e., formation of tetraploid pollen monads and diploid pollen dyads, and this is, in fact, what is observed after a colchicine treatment. However an important difference with respect to N20 treatment is that with colchicine it is impossible to treat the first division of meiosis without affecting also the second division simply because colchicine after penetrating in the cell takes a long time to bs dilutéd to a non-active levei.