Studies of meiosis in vitro

DEVELOPMENTAL

BIOLOGY

16, 36-53

Studies I. In Vitro MICHIO

( 1967)

of Meiosis Culture ITO’

of

in Vitro Meiotic

AND IIERBERT

Depurtment of Biology, Unioersity L.a Jollu, California

Cells1

STERN

of Californiu, Y2037

San Diego,

Accepted December 8, 1966 INTRODUCTION

The value of culturing meiotic cells in vitro is self-evident, and several attempts have been made to do so. The meiocytes of liliaceous plants are among the most attractive targets for in vitro culture because of their relative abundance, their synchronous development, and their protracted period of division (Erickson, 1948; Taylor and McMaster, 1954). A few investigators have had at least partial success in culturing intact anthers, but such organ cultures still present a number of obstacles to physiological studies, the most noteworthy one being the metabolic activity of the tapetal cells which envelop the microsporocytes. Attempts to culture isolated meiotic cells have proved to be abortive, and various reasons have been given to account for the failure. The two factors most prominently mentioned are the essential role of the tapetum and the syncytial organization of the meiocytes during early development. In one report emphasis has been placed on the progressive changes in the microsporocyte environment generated by the somatic tissues of the anther (Pereira and Linskens, 1963). In another report attention has been focused on the “damage inflicted during extraction” to the syncytium of microsporocytes and “which is likely to be propagated to all its parts, so that subsequent survival of any of its cells in vitro is unlikely” ( Heslop-Harrison, 1966). In this first communication we describe and discuss techniques which have been used in this laboratory for culturing meiotic cells of ‘This work was supported by a grant from the National Science Foundation ( GB-3902 ) . *Present address: Department of Biology, Nagoya University, Nagoya, Japan. 36

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Lilium and Trillium. Although the explanations offered for past failures do indeed have validity, the fact remains that meiotic cells have now been cultured through the meiotic cycle. Certain types of abnormalities do arise in culture, but these have not destroyed the suitability of the technique for studies of meiotic physiology. This will be apparent in Part II of this series (Ito et al., 1967), which is concerned with the behavior of meiotic cells beginning at the leptotene stage. Experiments on the physiological properties of premeiotic cells and their entry into meiosis under in vitro conditions will be published separately. METHODS

Media Lily microsporocytes. The basic culture medium was a White’s modified solution (White, 1963) containing the following components (in grams per liter) : Ca(NO&4 Hz0 KNOa KC1 MgSOa.7 Hz0 Na2S04 NaH2POcHxO

0.3 0.08 0.065 0.75 0.2 0.019

MnS044 Hz0 ZnSOA.7 Hz0 &Boa KI cuso4 NaaMoOa

5 3 15 75

x x x x

10-a 10-a 10-d 10-b 1 x 10-s 1 x 10-e

Fez(SO& glycine nicotinic acid thiamine pyridoxine

0.001 0.003 5 X lo-* 1 x 10-4 1 x 104

Sucrose was added to a concentration of 0.3 M and the pH was adjusted to 565.8 with a glass electrode. The medium was autoclaved for 15 minutes at 20 pounds pressure, and allowed to stand for at least 4 hours before being used. This standing was necessary because the pH of the medium drops during autoclaving and does not return to its original value in less than 4 hours. Usually, media were kept in the cold room overnight before being used for cultures. For certain experiments the following amino acids were added to the medium (each at 50 mg/liter): lysine, methionine, threonine, valine, isoleucine, leucine, glutamine, and glycine. Solid media were prepared by adding agar to a final concentration of 0.7%. The range of permissible sucrose concentrations and of pH were examined in some detail. Microsporocytes were cultured successfully in concentrations of sucrose between 0.25 and 0.35 ll4. Most other carbohydrates, especially monosaccharides, are toxic to the cells. The

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range of pH in which normal development occurs is between 5.4 and 6.2. Cytological abnormalities invariably arose in cells cultured outside these limits. Trillium microsporocytes. Media were prepared in the same way as for lily except that the basic medium was supplemented with 0.05-0.1% yeast extract and 0.35 M sucrose. Explantation Lily microsporocytes. Buds of desired length were collected from plants grown in a greenhouse. In all the species used for these experiments, bud length is correlated with meiotic development (Fig, 2). Species which lack this correlation are impractical for biochemical studies. The relationship between length of bud and meiotic stage varies slightly with temperature ( l-2 mm). Periodic checks at different seasons are recommended. After collection, the buds were kept moist until processed for dissection. The interval between collection and processing was rarely longer than 2 hours. Intact buds were sterilized by immersion in 70% ethanol for 1 minute and then dried with filter paper. All materials used following alcohol immersion were previously sterilized. For small-scale culture, the buds were opened and individual anthers were removed. Each anther was cut open at one end and the microsporocytes were extruded by gentle squeezing from the end distal to the cut. The microsporocytes emerged at the open end of the anther either as a coherent filament or as a viscous suspension of free cells ( Fig. I ) . The extruded filamentous aggregates were picked up with a pair of fine forceps and transferred to an appropriate medium. For large-scale culture the bud was not dissected but was cut with a razor blade at a distance below the apex such that the tips of all the anthers were removed. The microsporocytes were extruded by squeezing the entire bud and were then transferred in the same manner as the cells from individual anthers. The technique for small-scale culture is the more efficient, but it is also much more laborious if 50-100 buds are to be processed in one operation. Liquid cultures were carried out in 50 ml Erlenmeyer flasks containing IO ml of medium and plugged with absorbent cotton. At stages in which the microsporocytes emerge as filaments, 4 such filaments, about 4-6 mm in length, are obtained from each anther. No more than 120 filaments could be successfully cultured in a single flask. Solid cultures were carried out in g-cm petri dishes, each containing 20 ml of agar

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FIG. 1. The culture of meiotic cells. (A) Filaments of microsporocytes in a liquid culture medium. (B ) L ow p ower magnification ( X 60) of a filament of microsporocytes at leptonema after staining with propionic orcein. medium. All cultures were maintained without shaking at 20 1 1°C. Microsporocytes which were extruded as free cells at stages prior to metaphase I could be cultured successfully, if at all, only on solid media. At stages later than metaphase I free cells could be cultured in liquid media (Fig. 3).

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Trillium microsporocytes. The handling of Trillium plants and their seasonal behavior have been described in several publications (Sparrow and Sparrow, 1949; Hotta and Stern, 1963). Flower buds were removed and immersed for 5 minutes in diluted Clorox (a commercial preparation of 5.25% sodium hypochlorite which was diluted 2.5fold with water). The buds were then rinsed twice with sterile distilled water. Microsporocytes were extruded from individual anthers as described for lily. Cultures were usually carried out on solid media at 15°C. Contamination Microbial contamination of cultures occurred infrequently. Such contamination was clearly evident to the naked eye after S-4 days of growth. Generally, if cells were to be examined after shorter periods of time, samples were removed from the media and the remainder was allowed to stand for several more days. Since cytological examinations were made on all preparations, lower levels of contamination were automatically checked for microscopically. RESULTS

Factors Znfluencing

Survival

of Microsporocytes

in Culture

The respective problems of survival and development are best treated under separate headings. Microsporocytes which die during the period of culture do so within a few hours or less after explantation. Most such cells die within the first hour of culture, and even in cultures which have been maintained for 25-30 days, the number of dead cells found after 24 hours does not change significantly throughout the ensuing period. On the other hand, the occurrence of meiotic abnormalities among surviving cells is not correlated with time in culture but is a function of the particular meiotic process affected. Abnormalities would appear to be due to specific deficiencies in the medium which are related to the meiotic stage at which the cells are explanted. In general, the later the stage of explantation, the fewer are the abnormalities. This is not so with respect to survival. Cells which die when explanted at a particular stage of meiosis may complete meiosis if explanted earlier. The problem of survival appears to be related to incidental factors which vary from species to species and which lead to a rapid injury of cells when explanted into artificial media.

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On the whole, microsporocytes which can be explanted as coherent filaments survive well in culture (Fig. 1). Generally, filaments behave in one of two ways when placed in culture. Most of the cells within a filament either die quickly or they survive for the entire culture period. Frequently a small patch of living cells is found in the center of an otherwise dead filament and a small patch of dead cells (5-10%) is found at the ends of an otherwise living filament. In only a few cases are living or dead cells found to be scattered among their opposite types. Thus, to a certain extent, the claim that the syncytial organization of microsporocytes makes them extremely susceptible to injury is correct. Clearly, however, the wave of injury which has been viewed as moving through the entire filament from its point of origin (HeslopHarrison, 1966) can be sealed off. An approximate measure has been used to express the number of surviving cells in a culture. Filaments with 70% or more of living cells were counted as alive, and those with less than 70% living were counted as dead. Survival is expressed as a percentage of living over total filaments. Viability is easily detected either by the naked eye or through a dissection microscope. Dead portions of a filament are more opaque than living ones. This difference makes the removal of dead cells an easy task, and one which is particularly useful in biochemical experiments. These procedures of counting and separation cannot, of course, be used with freely suspended cells. For such cells individual counts had to be made and cytological criteria had to be used. The dead cells invariably showed shrunken nuclei which could be seen either directly with a phase microscope or after staining with propionic orcein. Microsporocytes obtained from Trillium erectum and from several varieties of lily were tested for their survival in culture with respect to the meiotic stage at which they were explanted (Fig. 2). The results showed marked and reproducible differences between species and between meiotic stages. However, microsporocytes from different species do not behave in parallel fashion with respect to stage of explantation. The meiotic cells of Croesus lilies, for example, are most susceptible to injury when explanted at leptonema whereas those of Georgia lilies are most susceptible at zygonema. Microsporocytes of Cinnabar are equally and highly viable at all the stages shown. From a practical standpoint, the suitability of Cinnabar for culture would appear to make further considerations superfluous, but the factors

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which are known to affect viability provide criteria species may be tested. Moreover, an understanding makes it possible to distinguish between properties incidental or intrinsic to the meiotic process.

by which

other

of these factors which

are either

100 60

m m m ix

e A m I 9.5

I IO

1 9 I 9

I II

I IO I II

12

12 I 13

I 12

II

1 14

I 13 1 15

A

13 /r\ 14

I lb

H~rmcmy IN, cro**u* Cinnobor

,CR, ICNI

FIG. 2. The survival in culture of microsporocytes from different species of lily and from Trillium. The methods used in calculating survival are described in the text. Survival is plotted against the stage at which the microsporocytes were erplunted. No relationship has been found between survival as plotted here and the viability of microsporocytes in culture at a particular stage if explanted earlier. Thus, microsporocytes of Trillium explanted at leptonema have a much higher proportion of living cells when they progress through zygonema than those which are explanted at zygonema. The lengths of the flower buds (mm) corresponding to the different meiotic stages are shown for each of the lily varieties tested. Croft and Nellie White are varieties of LiZium Zongiflorum. Harmony, Croesus, and Cinnabar are hybrids obtained from the Oregon Bulb Farms (Gresham, Oregon). Circled numbers (or circled points in the case of TriZZizrm) indicate stages at which the microspomcytes are extruded as free cells. Numbers in parentheses indicate stages at which either filaments or free cells are extruded. Numbers enclosed by triangles are stages at which microsporocytes are difficult anthers. Microsporocytes may be extruded as coherent filaments

to squeeze out of at all other stages.

Tapetal layer. This layer of cells which envelops the microsporocytes in liliaceous plants, and for which a variety of functions has been postulated, undergoes degeneration at some time prior to pollen matu-

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ration. However, the developmental stage at which this occurs and the firmness of attachment between tapetum and adjacent cell layers varies from species to species. At stages in which the attachment is firm, microsporocytes not only are difficult to squeeze out of the anther, but those which are extruded survive poorly in culture. Presumably, the cells are injured by the pressure of extrusion. The conclusion may be drawn that a loose attachment between tapetum and microsporocyte mass is a necessary condition for in vitro culture, but it is not a sufficient one. The poor survival of leptonema cells from Georgia or Croesus lilies and the relatively good survival of those from Cinnabar or Nellie White correlate with this property of attachment. On the other hand, as shown in Fig. 2, there are large differences between species even when their microsporocytes mav be easily extruded from the anthers.

tLept.+Zyg.e

FIG. 3.

Survival

of microsporocytrs

Pach.HDipl

(Nellie

-Diak.+I

White)

B II+

in culture

as coherent

filaments or as free cells. Solid circles indicate cultures on solid media; open circles indicate cultures in liquid media. The conditions under which pachytene cells may be obtained either as filaments or as free cells are discussed in the text. Other details are the same as under Fig. 2. The reader should again note that the stages refer only to the time of explantation.

Coherence of filaments. Microsporocytes which are easily extruded during early meiosis usually emerge as coherent filaments. Generally, coherent filaments survive well in culture; free cells do not. In Trillium, the drop in survival rate from leptonema to zygonema correlates with the disappearance of cohesiveness among the microsporocytes at the later stage. This species is nevertheless exceptional in that even the free cells show a relatively high degree of survival in culture. How the emergence of microsporocytes as coherent filaments relates to their functional organization in situ is not entirely clear. Pachytene cells extruded from lilies grown at optimal temperatures (about 22°C)

FIG. 4. Cytological appearance of cultured I’hese cells (var. Nellie White) were explanted 44

cells at various meiotic stages. at zygonema. The photographs

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emerge as filaments, whereas those extruded from plants grown at higher temperatures emerge as free cells. Microsporocytes within the plants developed normally under both conditions. If explanted, however, those emerging as free cells survived poorly in culture (Fig. 3). The coherence of microsporocytes at pachynema is thus an incidental property which has an important bearing on meiosis in vitro but not on meiosis in situ. Indeed, for practical purposes, plants which have been growing at undesirably high temperatures may be retrieved for culture experiments if brought to lower temperatures several days before use. Meiotic stage. Superimposed on the factors already discussed, are others which are specifically related to meiotic stage. Cells explanted at the prophase of meiosis are more susceptible to injury than those explanted at metaphase I or later. Free microsporocytes survive well after metaphase I (Fig. 3). Three general observations may be made on these studies. First, various conditions of tissue organization exist within anthers of different species, and these may facilitate or hinder the culture of microsporocytes in vitro. These conditions are considered to be trivial inasmuch as they do not reflect any intrinsic and universal properties of the meiotic cells themselves. Although methods could probably be devised to overcome the adverse effects of these conditions, the most practical approach is to use suitable species. Second, some form of physical association between the microsporocytes appears to be essential to their maintenance in culture. One factor in this association is probably the syncytial organization of the microsporocyte mass. This cannot be the sole factor, however, because the need for close packing in vitro persists even when the syncytial organization in situ has. disappeared. Here too, the choice of a suitable species appears to be the most practical solution to the problem. Third, the nutritional conditions permitting survival of microsporocytes in vitro appear to be simple illustrate the loss of synchrony which occurs under conditions of culture. This point is discussed in the text. Individual stages are shown at higher magnification in Fig. 5. Occasional tapetal nuclei may be seen among the microsporocytes. These were judged to be inactive from autoradiographic studies. Labeled precursors of protein, RNA, or DNA were not incorporated by the nuclei, a property which is significant for biochemical studies. The walls of the tetrads (E, F) are appreciably thicker than those of cells developing in situ. Approximate magnif& tion: x 150 (except E which is x 120 ).

microsporocytes of F IG. 5. Cultured cells at various Phot’ ographs of individual illustrated in Fig. 4. from the preparations (E) metaphase (Cl and (D) diakinesis; magnification: X (H) tetrad. Approximate 46

Lilium Zongiflorzrm (Nellie White). meiotic stages. The cells were selected (A) Early pachynema; ( B ) diplonema; I; (F) anaphase I; (C) mrtaphase II; 640.

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although we have no information on the substances which are explanted together with the microsporocyte mass. However, we can clearly point to the importance of the most active osmotic agent in the medium, sucrose. A variety of osmotic agents has been tested for all periods of meiosis, but sucrose is by far the most satisfactory. All the simple monosaccharides we have tested (hexoses and riboses) are extremely toxic, causing death within several minutes. The toxicity persists even if sucrose is present in the medium. On the other hand, phosphorylated monosaccharides (ribose 5-phosphate, glucose 6-phosphate, fructose 1,Sdiphosphate) are not appreciably toxic. These results lead us to the conclusion that the free monosaccharides not only penetrate the meiotic cells, but are also incompatible with intracellular This incompatibility raises a number of physiological requirements. interesting questions, but as the following paper shows, our interests lie in a different direction.

Cytological Characteristics of Cultured Cells The ease with which living cells may be separated from dead ones and the availability of certain species of microsporocytes which survive when explanted at any meiotic stage, makes the factor of survival a minor one for physiological studies. The major question which must be raised concerning the culture technique is whether the meiotic cells develop normally in vitro. To this question the answer is unambiguous in two respects. First, microsporocytes explanted in leptonema show cytological abnormalities; those explanted at zygonema or later show very few (Figs. 4-6). From mid-pachynema on, the frequency of in abnormalities is the same in cultured cells as in those developing which develop due to cultursitu (Fig. 7). Second, the abnormalities ing are of one kind. All are in the nature of aberrant segregation. The only other type of abnormality noted is a failure of the second meiotic division, and this occurs almost exclusively in microsporocytes which have been cultured as free cells rather than as filaments. A point to be emphasized is that chromosomal events are normal up to the metaphase stage. Pairing, chiasma formation, and contraction are undisturbed; the spindle mechanism appears to be the source of abnormalities which are manifest at the conclusion of meiosis in the geometry of wall formation, and in the number and size of the nuclei within what is otherwise a symmetrical aggregate of 4 haploid cells. Thus, some factors which are essential to spindle organization are

FIG. 6. Meiotic abnormalities in cultured cells. (A) Microsporocytes of Nellie White explanted in late leptonema-early zygonema and cultured for 6 days. In some of the cells, the chromosomes are returning to an interphase configuration. The aberrant segregation pattern of the chromosomes is apparent in most cells. Magnification: x 300. ( B ) Microsporocytes of Nellie White explanted at the same stage as those in (A), but cultured in a medium supplemented with amino acids. 48

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disturbed if cells are explanted during leptonema. Whether certain preformed components are destroyed during explantation at leptonema or whether the explanted cells lose their capacity to form essential components is unknown. Various substances were tested for their possible effects on preserving the spindle mechanism. Among these, coconut milk, amino acids, or water extracts from the meristematic portions of roots proved to be effective in restoring normal spindle function to cells explanted in mid to late leptonema but not if the cells were explanted in early leptonema (Figs. 6 and 7). The hormonal substances, indoleacetic acid, gibberellic acid, kinetin or 2,Pdichlorophenoxyacetic acid, were tested over the range of 15 mg/liter. These either had no effect on meiosis or induced new abnormalities. These tests were made only on cells in coherent filaments. Thus, to the extent that the culture technique described is aimed at studying the behavior of cells from pre- or early meiosis to its completion without any abnormality in chromosome function, it remains deficient. On the other hand, if studies requiring normal chromosome segregation may be started at zygonema the culture technique is adequate. For studies of pairing and chiasma formation, two distinctive meiotic events, the technique is now adequate if certain species of plants are used (see Fig. 6 for a comparison between Nellie White and Cinnabar microsporocytes ) . Synchrony and Duration

of Meiosis in Cultured Cells

One noticeable effect of in vitro culture on meiotic cells is the accelerated loss of synchrony. Generally, under in situ conditions, the suitable varieties tested maintain a high degree of synchrony up to diakinesis-metaphase. Beyond these stages synchrony begins to be lost. In culture, however, a spread in stages is quite apparent around diplonema. Data on loss of synchrony and also on rates of meiosis in culture are shown in Figs. 8 and 9. Under greenhouse conditions The cells shown in this photograph were fixed after 6 days of culture. The beneficial effects of amino acids on spindle function are discussed in the text. Magnification: x 150. (C) Microsporocytes of Cinnabar explanted at late leptonema-early zygonema and cultured for 6 days in basic medium without amino acid supplement. In contrast to Nellie White cells, these show relatively few segregation abnormalities. Magnification: x 120. (D) Tetrads of Cinnabar microsporocytes explanted in mid-zygonema and cultured in basic medium for 6 days. Magnification: X 280.

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I ’

ZYW.

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1 ’

Path.

I ’ DipI.-Diak.

, ‘I

EI

FIG. 7. Meiotic development of microsporocytes (Nellie White) cultured in uitro. Ordinates represent the percentage of normal meiotic products over total number of living cells. Cells which showed the typical tetrad configuration were counted as normal. Stages refer to the times at which the microsporocytes were explanted. In all cases, cells were allowed to remain in culture until meiosis was complete. Filaments were cultured in liquid media; free cells were cultured on solid media. The curve marked AA is for cultures in which the medium was supplemented with amino acids as outlined under Methods. Those supplemented with 15% (v/v) coconut milk are marked CM. The beneficial effect of these supplements on spindle function is discussed in the text.

Tet. c Am-II M-n Inter. A-I M-1 Diak. Dipl.

L0pt.

( 14) (!f=

I234

5

6

7

8

9

IO

II

12

DAYS FIG. 8. Rates of meiosis in cultured microsporocytes of L&urn Zongijbrum (Nellie White). Microsporocytes were explanted at day 0. Meiotic stages are marked on the ordinates to indicate the stages of the cells either at the time of explantation or in culture on the day examined. Bud lengths are shown in parentheses for those stages at which cells were explanted. The bars span all the stages found in a particular preparation. This representation of meiotic development does not discriminate between normal and abnormal chromosome segregation. The progress of Trillium microsporocytes in culture is shown by the dotted line.

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(about 25%) microsporocytes of lily (Nellie White) take about 7-8 days to complete meiosis from the leptotene stage; in culture at 22°C the interval is about S-9 days. The rates are thus essentially the same. Trillium microsporocytes develop more slowly in culture but are exposed to a lower temperature, 15°C. This rate is difficult to compare with the in situ one, because Trillium plants are generally stored at about 3°C and the microsporocytes undergo meiosis during storage. We have not tested cultures at 23°C.

of meiotic stages in cultured microsporocytes (Nellie FIG. 9. Distribution White). Twenty-four filaments of microsporocytes were explanted from a single bud (16 mm) and cultured in a single flask. The cells were all in zygonema. On the second day of culture and on succeeding days, four filaments were removed for cytological analysis. The percentage of meiotic stages in a preparation after a given period of culture is shown by the curves. The number above each of the curves indicates the number of days in culture. For comparison, the distribution of stages in the microsporocytes of an anther developing in situ and removed at a bud length of 23 mm is shown by the dotted line. The lower degree of synchrony in cultured microsporocytes is made apparent by this comparison. DISCUSSION

The usefulness of the methods described for culturing meiotic cells will become apparent in the application of the technique for the solution of specific meiotic problems. The experiments themselves, however, do throw some light on certain developmental aspects of microsporogenesis. The very high susceptibility of microsporocytes to rapid injury is a property rarely encountered in other plant cells, and it undoubtedly reflects the unique physical organization of the microsporogenous tissue. That such physical organization has a direct relevance to meiosis itself is, nevertheless, unlikely. Most probably, the organization is an adaptation to the special conditions under which microsporogenesis occurs in higher plants. Thus, from the standpoint

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of those who are primarily interested in the process of meiosis alone, the difficulties encountered in studies of liliaceous plants can be justified only on the basis of their special value for physiological investigations. From the standpoint of those interested in tissue relationships, the results provide some answers and raise as many questions. The behavior of microsporocytes in culture is consistent with the view that the role of the tapetum is to provide a permissive environment for meiotic development. It is inconsistent with the view that the tapetum plays a direct role in inducing specific meiotic developments. Our conclusion is thus in line with the views of Takats (1959), who provided evidence against macromolecular transfers between somatic and sporogenous tissues. The culture experiments nevertheless do not define the nature of the “permissive environment” generated by the tapetal tissue. This deficiency is made clear by our failure to culture microsporocytes from early leptonema through the cycle without introducing abnormalities in chromosome segregation. Moreover, our studies have in no way encompassed the mechanisms by which the original cell population of the developing anther differentiates into distinctive layers of meiotic and mitotic tissues. SUMMARY

: A method has been described for the in vitro culture of meiotic cells derived from various species of liliaceous plants. Only some of the species tested are suitable for the method. Using such species, meiotic cells may be explanted at zygonema and cultured through the meiotic cycle. If explanted at leptonema,- segregation abnormalities arise but synapsis and crossing over remain normal. REFERENCES

ERICKSON, R. 0. ( 1948).

Cytological and growth correlations in the flower bud and anther of Lilium longijlorum. Am. J. Botany 35, 729-739. HESLOP-HARRISON,J. ( 1966). Cytoplasmic continuities during spore formation in flowering plants. Endeavour 25, 65-72. Ho~A, Y., and STERN, H. ( 1963). Inhibition of protein syntheses during meiosis and its bearing on intracellular regulation. .J. Cell Biol. 16, 259-279. ITO, M., HOTTA, Y., and STERN, H. (1967). Studies of meiosis in uitro. II. Effect of inhibiting DNA synthesis during meiotic prophase on chromosome structure and behavior. Develop. Biol. 16, 54-77. PEREIRA, A. S. R., and, LINSKENS, H. F. ( 1963). The influence of glutathione and glutathione antagonists on meiosis in excised anthers of Lilium henryi. Acta Bothn. Need. 12, 302-314.

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A. II.,

to and during

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and SPARROW, R. C. (1949). Treatment of Trillium erectum prior mass production of permanent smear preparations. Stain Tcchnol.

24,475s. S. T. ( 1959). Chromatin extrusion and DNA transfer during microsporogenesis. Chromosoma 10, 430-453. TAYLOR, J. II., and MCMASTER, R. ( 1954). Autoradiographic and microphotometric studies of deoxyribose nucleic acid during microgametogenesis in L&urn Zongiflorum. Chromosoma 6,489-521. WHITE, P. R. ( 1963). “The Cultivation of Animal and Plant Cells” (2nd ed. ). Ronald Press, New York. TAKATS,