Plant Tissue Culture in Relation to Developmental Cytology

Plant Tissue Culture in Relation to Developmental Cytology CARLR. PARTANEN Department of Biologiial Siienies, L'niversit)J

of

Pittsburgh, Pittsburgh, Pennsjlvar2ia Page

. . . . . . . . . . . . . . I. Introduction . . 11. Plant Tissue Culture . . . . . . . . . . . . . . . . . . . . A. Definition . . . ....................... B. Methods of Plant Tissue Culture . . . . . . . . . . . . . . . . . . . 111. Cytology in Viva . . . . . . . . . . . . . . . . . . . . . . . . . . ................ IV. Cell Differentiation . . . . . . . V. Cytology in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . €3. Aneuploidy . . . . . . . . . . . . . . . . . . . . . . . . . . C. Haploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Cytological Technique . . . . . . . . . . . . . . . . . ..... VII. Morphogenetic Studies . . . . . . . . . . . . . . . . . . . A. Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . . . B. Cell Cultures . . . . . . . . . . . . . . . . . . . . . . VlII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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I. Introduction Plant tissue culture was put into its proper perspective long before it was achieved (Haberlandt, cf. White, 1943). It was the desire to understand plant morphogenesis and the recognition that developmental processes are basically cellular processes that led to the recognized need for studying living cells apart from the organism. And now, approximately a quarter century after the technique of culturing plant tissue in vitro was first attained, the original goals are gradually being realized. The intervening years have been involved in a myriad of technical problems of varying degrees of complexity. Indeed, some of these tributary problems have in themselves become so involved that the main stream may at times have seemed difficult to define. Of course, plant tissue culture as a tool has found applications that were perhaps not originally envisioned, and which do not pretend to be aimed toward the original long range goals. At times the technique appears to have become an end in itself. However, there has also been a continuing interest in the application of plant tissue culture to problems of morphogenesis and cellular differentiation. It is with this application, primarily in relation to nuclear cytology, that this present consideration will be concerned. This will not attempt to be a complete review of plant tissue culture and those areas of cytology that are touched upon. Extensive reviews are available for a more complete treatment of the separate areas (Gautheret, 1959; D'Amato, 1952; Steward et al., 1961; Tulecke, 1961; 215

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Braun and Stonier, 1958). Instead, the attempt here will be to bring certain observations into meaningful relationships.

11. Plant Tissue Culture A. DEFINITION The maintenance and growth of plant cells in vitro under aseptic conditions constitutes plant tissue culture. The cells may be of various types and in varying degrees of association. The characteristic cell type, regardless of the tissue of origin, is usually recognizable as being similar to that which in wiwo is called parenchyma. However, there is generally some degree of heterogeneity among the cells constituting such a culture or callus, to the extent that certain cells may undergo visibly recognizable differentiation, most frequently to a cell type resembling a xylem element, although functionally they do not comprise xylem because they usually occur singly. Other cellular variations also are found with regard to cell size, shape, distribution of accumulation products, and pigments. The cell aggregate can be considered a “tissue” in a loose application of a definition of the term: “material formed by the union of cells, which may be similar in character (simple tissue) or varied (complex tissue) ” (Esau, 1960). Thus, plant tissue cultures generally may be considered to be tissues intergrading from “simple” to “complex.” These are purely descriptive concepts with no functional implications. Further difficulty in the application of the term arises when one considers the cultures in which the cells are essentially dissociated, i.e., free cells, as in some of the cultures which are maintained in liquid, with agitation. These, perhaps, should more properly be termed “cell cultures.” However, for purposes of our present consideration, and with the above reservations and qualifications, we shall include under the broad category “tissue culture” all growth of plant cells in vitro other than organized systems such as roots, leaves, or intact plants. In addition to the culture of plant tissues, the general technique also lends itself to the aseptic culture of plants and plant parts. For example, the various aspects of the life cycle of ferns have been extensively investigated by the application of this approach: sporophytes (Wetmore, 1954) ; detached leaves (Steeves and Sussex, 1957; Sussex and Steeves, 1958) ; roots (Partanen, unpublished) ; gametophytes and gametophyte tumors (Steeves et a/., 1955 ; Partanen et al., 1955) ; embryogeny (DeMaggio and Wetmore, 1961) ; apogamy (Whittier and Steeves, 1960). There have been many other investigators and studies in this area. Although these are sometimes included in the general category “plant tissue culture,” this categorization is simply a matter of reference to the techniques used. Therefore, these studies will be largely omitted from this discussion, except where they are relevant, e.g., tissue proliferations derived from such plants or plant parts and cultured separately.

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B. METHODSOF PLANTTISSUE CULTURE The initiation of a plant tissue culture usually involves the excision of a portion of a plant (other than an apical meristem or a bud) and placing this explant on or in a suitable substrate under aseptic conditions. Various portions of the plant may be used, e.g., stem pieces, including tuber and rhizome, either whole sections or small pieces, depending upon the size and nature of the stem, In large woody stems, for example, cambial explants can be used. Other commonly used sources of explants are sections of root or various portions of embryos, such as hypocotyl. In some cases the intact embryo will readily produce an overgrowth of callus tissue which can be isolated and cultured separately. (For a comprehensive review of plant tissue culture techniques see Gautheret, 1959.) Rarely, such sources as pollen can be induced to produce a tissuelike proliferation (Tulecke, 1957). Also, tumors which are “spontaneous,” “genetic,” or “crown gall” are capable of being cultured in vitro. These often involve a recognized causal factor which may be endogenous, e.g., genetic imbalance in certain hybrids, or exogenous, e.g., viral, bacterial (Braun and Stonier, 1958), or physical, e.g., ionizing or ultraviolet radiations (Partanen and Steeves, 1956; Partanen and Nelson, 1961). The composition of media may vary considerably. A basic medium contains one of a number of mineral salt solutions which contain both major and trace elements (cf. Gautheret, 1959). For a very few tissues this will suffice, e g . , tumor tissue capable of photosynthesis, although the growth of even such tissues is enhanced by the addition of a small amount of sugar (Partanen et ul., 1955). As the spectrum of tissues goes toward those with less capacity for the synthesis of essentials for cell division and growth, the complexity of the medium increases. In nonphotosynthetic tissues a higher concentration of sugar is optimal. Most tissues are dependent upon the addition of a plant growth hormone (auxin) such as indoleacetic acid or one of its effective analogs. There are certain notable exceptions to this requirement, primarily among the tumorous tissues and those which have become “habituated” in culture. In some tissues, notably tobacco pith, it has been shown that another endogenous factor in addition to auxin is limiting to the process of cell division. This requirement can be met by adding kinetin, one of the kinins (Skoog and Miller, 1957). Various other requirements for growth of certain specific tissues have been defined including such additives as vitamins and amino acids. To this extent the media can be considered “chemically defined,” although if the medium is solidified with agar, that undefined component should not be overlooked in the final analysis. Many tissues will not grow satisfactorily, or at all, on present chemically defined media. In these cases growth is often achieved by the addition of such complex substances as the liquid endosperm of coconut (coconut “milk’ or “water”) or extracts of it, or other endosperms (van Overbeek et ul., 1941; Caplin and

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Steward, 1948; Tulecke et al., 1961). This has enabled the culture of many otherwise totally or relatively unresponsive tissues. However, for purposes of many types of experimentation the addition of such complex factors is undesirable, because they unduly complicate the problem of interpretation. Not-

FIG. 1. Pings lambertiana in vitro. A. Callus grown on agar medium, x 13; B. CLusters of cells dissociated from callus grown on agar, ca. x 200; C-F. Free cells from liquid culture of pine grown in spinner flask. ca. x 200.

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ably refractory to being cultured satisfactorily are the tissues of monocotyledonous plants, among which are to be found, unfortunately, some of the best materials for chromosomal studies. Methods of plant tissue culture vary somewhat from one laboratory to another with respect to such factors as the environment in which the cultures are kept and the nature of the culture vessel and the substrate. Media may be either solid or liquid. On solid media the culture usually grows as a single mass of callus tissue which may vary in character from very coherent, hard tissue to a rather friable or “mushy” association of ceJls. Solid media are generally used in culture tubes, flasks, or bottles which are closed with cotton plugs, plastic film, metal foil, or solid caps. The use of liquid media generally involves some provision for aeration. The tissues may be held above the surface of the medium by various devices, or if submerged in the medium may be aerated by physical movement of the entire vessel as on a shake table, roller drum, or similar device. Aeration and agitation may also be accomplished by passing a stream of sterile air into the medium, or by growing the culture in a spinner flask. The culturing of tissues in a liquid which is being agitated in some manner often results in a looser association of cells which may dissociate into free cells and small groups of cells. Examples of plant tissue and cell cultures are shown in Fig. 1. Plant tissue cultures, once having been attained, have become the subject of many types of investigations. Many have become a perpetuation of the technique itself in the elaboration of better growing conditions, primarily the definition of media which will produce greater growth rates. On the one hand, this approach furthers the ends of all who work with tissue cultures in defining the requirements of such tissues for rapid proliferation, but on the other hand, if an understanding of morphogenetic processes be one of the desired goals, this may unwittingly be working in another direction. That is, the totality of morphogenesis consists not only of division and growth of cells, but also of the cessation of these processes and the initiation of biochemical and morphological changes of cells in an organized system, i.e., cell differentiation. An avenue of approach toward the morphogenetic ends has been the morphological, anatomical, and cytological characterization of plant tissue cultures, and in some cases the correlation of such observations with the prevailing conditions. This discussion will be concerned with the cytological aspects, primarily nuclear, of plant tissue cultures, and some implications of these observations.

111. Cytology in V i m Before discussing chromosomal behavior it2 v i ~ i o ,a proper point of departure would be a brief consideration of the chromosomes of plants in viuo. Plant species are of course characterized by a constancy of caryotype with respect to

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chromosome number and morphology. This is evident if the chromosomes are examined in dividing cells of apical meristems, shoot or root, as well as in numerous other places in which divisions are occurring in the growth of the plant. In all of these the characteristic diploid chromosomal complement is usually seen. And, by examining the gametic chromosomes, the haploid number is almost invariably encountered. The exceptions in these cases are relatively infrequent. To this extent, the concept of caryotype constancy is nearly absolute. However, during the past quarter-century another concept, that of somatic chromosomal inconstancy, has evolved. In brief, numerous observations of chromosome number, primarily in nonmeristematic portions of plants, have led to the knowledge that the chromosome number may vary among the cells of an individual plant (for reviews cf. D’Amato, 1952; Geitler, 1953). By far the most common variation is an increase in chromosomal material which, if the nucleus is to divide, manifests itself as an increased chromosome number. An increase can also appear in another way, by the presence of more than one nucleus per cell. These increases in chromosomal material can, in effect, all be considered to arise in essentially the same way, i.e., through a breakdown of the usual sequence of processes involved in cell multiplication: replication, nuclear division, and cell division. That is, although chromosomal replication is, in meristematic regions, followed by mitosis, and that in turn by caryokinesis, they need not necessarily do so in all somatic cells. From such observations it is self-evident that these are separate and separable processes. If, upon completion of replication of the chromosomes the nucleus does not enter mitosis, there are at least two alternatives: either it can remain at that level or it can continue replication. In the latter case, all existing units are duplicated. In many cases this cycle may be repeated more than once. Hence, the progression in chromosomal units through repeated cycles is a geometric one. The amount of deoxyribonucleic acid (DNA) per completed chromatid set being constant (Alfert and Swift, 1953), this condition produced by additional somatic nuclear replications lends itself to quantitative analysis by the measurement of D N A in individual nuclei by cytophotometric estimation of the Feulgen reaction (Swift, 1953 ; Pollister and Ornstein, 1955). The unduplicated diploid level of D N A is designated “2C.” The geometric progression therefore reflects itself in a population of such somatic nuclei with peak values at 2C, 4C, sC, lGC, etc. There are certain distinctions that the technique cannot make, e.g., an 8C interphase nucleus can either be a duplicated tetraploid or unduplicated octaploid. However, in either case it is reflective of at least that amount of prior synthesis having occurred. If the increase in chromosomal material occurred by a breakdown of the sequence between replication and mitosis, there is usually another criterion evident during the first nuclear division after such an additional replication cycle.

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The chromosomes have double the usual number of visible chromatids (diplochromosomes) after one additional cycle, four (quadruplochromosomes) after two, etc. The resultant division yields two nuclei with an increased chromosome number. This process of additional replications may occur with relatively few if any visible manifestations during the prolonged interphase, or it may be marked by visible events within the nuclear membrane, including a contraction of resolvable chromosomes. This latter case was called “endomitosis” by Geitler (1’939). However, the numerous subsequent observations on additional interphase nuclear replication cycles in various species suggest a range of types with respect to the concomitant visible events. This has given rise to a general confusion, disagreement, and multiplicity of terminology (cf. Geitler, 1939, 1953; D’Amato, 1952, 1954; Levan and Hauschka, 1953; Partanen, 1959, 1961b; Rasch et al., 1959). However, with the realization that there are species differences in the degree of the visible manifestation and also that some of the distinguishing characteristics may in some cases involve problems of optical resolution, it appears that all are essentially the same process. With that broad view, it is felt that no ambiguity, but rather conceptual clarity, is obtained by using the original term “endomitosis” for the process of an additional chromosomal replication during an interphase, which may vary in its manifestations to approximately the same extent as do many other biological processes observed in a number of species. This may be countered with the objection that “endomitosis” is a morphological concept (Rasch ef al., 1959). However, that is precisely the point; intracellular morphology does not cease to exist at some convenient arbitrarily chosen visual limit. The consequences of endomitosis are evident spontaneously in some species of plants, but more often can be demonstrated by treatment with a high concentration of an auxin (cf. D’Amato, 1952) which causes mitosis in various mature cells. The results of such studies show that somatic polyploidy (polysomaty, endopolyploidy) is a relatively widespread phenomenon in many types of tissues examined. It occurs in cells which are “differentiated” (cf. Partanen, 1959, 1961a, b ) , i.e., nonmeristematic, using “meristematic” to refer to those groups of cells which continue to produce new cells and which are self-perpetuating during the active growth of the plant. In brief, endopolyploidy has been found in almost every conceivable type of plant tissue, among the various species. However, it does not, apparently, occur in all species, nor in all tissues of those which do display it, nor even necessarily in all cells of such tissues. Instead, it is frequently scattered in its distribution, though various tissues may have a characteristic distribution of different levels of ploidy. The morphogenetic implications of this phenomenon have been the subject of considerable discussion (cf. Huskins, 1948, 1952 ; D’Amato, 1952; Partanen, 1959, 1961a, b ; Torrey, 1959). The obvious concomitance of somatic polyploidy with cellular differentiation has led to varying views concerning the

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possible relationship between the two phenomena. But most analyses show that polyploidy is a variable concomitant of cellular differentiation. Entire systems can apparently differentiate without it, and where it does occur, it can vary in degree and frequency (Partanen, 1961a). To place the phenomenon of endopolyploidy into its true perspective requires a consideration of how it may arise. In the meristematic regions of an intact plant, where the basic chromosome number prevails, the processes of nuclear replication, mitosis, and caryokinesis, are rather closely coupled or sequential. That is, each preceding step seemingly insures the following one. Or, to state it another way, apparently the only factor limiting any step is the satisfactory execution of the preceding one. Undoubtedly, numerous factors are contributory to the culmination of each step, such as the availability of the necessary precursors or components of the structural elements involved, and the energy required to assemble or operate them. An appreciation of the complexity of the factors involved (cf. Stern, 1960) emphasizes once again the intricacy of an integrated, functioning biological system. These nuclear events must be very intimately tied into the total metabolism of the cells. That these separate processes involved in cell multiplication do operate in such close harmony would indicate that all of the contributing factors to each are optimally available in an active meristematic region. However, as a consequence of repeated divisions, some cells are left behind. Numerous locally varying chemical differences are demonstrable in plants; these may be due to differences in transport and localization of synthesis. Consequently, differences in position of cells with respect to the total system can mean differences in the chemical, as well as the physical, environments of such cells. Some of these varying factors may be directly or indirectly involved in one or more of the separate processes of cell multiplication. If, for example, conditions are suitable for chromosomal replication, but some factor for cell division is limiting, then the stage would be set for endomitosis, resulting in endopolyploidy (cf. Partanen et ul., 1955 ; Partanen, 1959, 196lb). Ordinarily these cells do not divide again; therefore their polyploidy is not detected. However, if the plant is treated with a high concentration of auxin, this, by some as yet undefined mechanism, induces these nuclei to divide. A basis for this type of chemical control can be seen in the work of Das et a/. (1956) and Patau et al. (1957), who showed that two endogenous factors, an auxin and a kinin, can both be limiting for the completed sequence of cell multiplication in excised tobacco pith. Presumably this can be taken as a model for other systems as well, although conceivably other factors may also become limiting. There is, however, always a danger in attributing specificity to the factor which happens to be limiting a particular phenomenon in a particular instance.

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IV. Cell Differentiation

As nuclei may reflect the cell’s changing environment within the plant body, so may other components of the cell. Eventually, some of these changes may show recognizable morphological differences. Undoubtedly any eventual morphological cellular difference has a preceding series of biochemical events. These would, in our interpretation, be included under the phenomenon of “differentiation.” Thus, there may be differentiation in which no recognizable morphological manifestation occurs, or is evident at the time of the analysis. Therefore, to attempt to correlate the nuclear changes with the general process of differentiation may be rather foolhardy. But, by the same reasoning, the presence of a changed nucleus can in itself be considered to be a type of differentiation. The question then becomes one of how various types of cell differentiation may be correlated. Probably no particular type of cell differentiation is a simple or single pattern of events. Thus, several types may be concomitant; which ones they are would seem to be largely a matter of which general metabolic patterns are affected or involved and what the eventual manifestations of these changes may be. To the extent that the behavior of the nucleus is affected by such events, the nuclear change can be a concomitant of whatever other change may also occur. Thus, endomitosis and/or the failure of cytokinesis can be looked upon as a type of cell differentiation which may or may not accompany another type. The extent to which certain specific types of differentiation are concomitant would seem to be a reflection of the extent to which common factors are involved. Thus, in this type of change, causality of the nuclear change to the other changes would be unlikely. Yet, polyploidy, once having been reached in a cell, may then have an effect upon the fate of that cell in a mixed population. It is not known if all processes are increased in proportion to the nuclear increase in such a cell, e.g., the rate of respiration. It would seem to be self-evident that such a cell may have increased demands, insofar as during the replication cycle there is more material to be synthesized. This may also involve a greater energy demand for the synthesis as well as for the subsequent events, mitosis and cytokinesis. There are some indications from cytophotometric studies of the degree and extent of endomitosis in a polyploid series of fern gametophytes that such considerations as these may indeed be involved (Partanen, 196lb). Thus, in this sense, somatic polyploidy may conceivably be the forerunner of yet other types of cell changes within the organism, but these possibilities remain to be observed and documented accurately. It is implicit that this concept of differentiation is valid only for the consideration of a particular cell in relation to the whole intact system in which it is changing. However, that is a realistic limitation since the cell does not differentiate alone, but rather in relation to the whole system. Alter the system, e.g.,

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by the introduction of exogenous agents or by excision of a part, and the cells are no longer in the same system in which they differentiated. They are then free to change to the extent that they are permitted by their own potentialities to respond to the altered conditions in vivo or in vitro. Hence, cells which we consider differentiated in the intact plant are capable of becoming proliferating masses of active parenchyma, behaving in a limited meristematic fashion it2 Z J ~ W U . Now, any subsequent differentiation of such cells i ~ zvitvo must occur within their new context, the tissue mass. This is not an organized system in the sense of the plant from which the cells came, the relationship to which and in which their genetic potentialities of the genome have evolved. Thus, the cells may not respond in a recognizably organized manner. Various manifestations may be sporadic, reflective of local conditions and the capacity of the prevailing genotype to respond to them, The initiation of an organized system is probably dependent upon some rather specific environmental conditions, at least up to a point, at which stage an established pattern may become self-perpetuating (cf. later discussion of morphogenetic studies).

v.

Cytology in Vitro A. POLYPLOIDY

One of the most commonly reported nuclear variations in both normal and tumor tissues grown in uitro is polyploidy. It has been observed in the following: “Normal” tissue: Daucus carota (carrot) Ginkgo bdoba (maidenhair tree, pollen)

Mitra et al. (1960) Tulecke (1957)

Loliurn perenne (rye grass, endosperm) Nicotiana Jabarum (tobacco) Nicotiana glutinosa Picea glauca (white spruce) Pinus lam bertiana (sugar pine) Pisum sativum (pea) Sequoia sempervirens Vicia faba (broad bean) Zea mays (corn, endosperm)

Mitra and Steward (1961) Reinert and Torrey (1961) Norstog (1956) Skoog (1954) Partanen (unpublished) de Torok and White (1960) Partanen (unpublished) Torrey (1959) Partanen (unpublished) Venketeswaran (1962) Straus (1954)

“Tumor” tissue: Osmunda rinnamomea (cinnamon fern) Picea glauca (white spruce) Ptevidium aquilinum (bracken fern)

Partanen et a / . (1955) de Torok and White (1960) Partanen et al. (1955)

Haplopappus gracilis

The observations of polyploidy in normal tissue cultures vary considerably in the extent of the supporting data presented. Some simply mention the phenomenon (Skoog, 1954). Some discuss it briefly (Norstog, 1956; Tulecke,

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1957). Others present no actual data, but discuss the observations in considerable detail and present photographic examples (Straus, 1954; Mitra et al., 1960; Mitra and Steward, 1961) ; while still others include considerable data in the form of tabulation of large samples of counts (Torrey, 1959; de Torok and White, 1960). Among the few types of tumor tissues in vitro that have been studied cytologically, polyploidy has appeared in all, i.e., fern gametophyte tumors, both spontaneous and radiation-induced (Partanen, 1956; Partanen et al., 1955) and spontaneous tumors of spruce (de Torok and White, 1960; de Torok and Roderick, 1962). The studies of Partanen (1956) included an analysis of the amounts of DNA in individual nuclei of normal gametophyte tissue, early isolated tumor, and established tumor. These showed quite clearly the relationship between increased chromosome number and the process of tumorization in this species. This study took into consideration the nondividing cells as well as those which were currently dividing. It showed that chromosome counts are not necessarily an accurate criterion of the general nuclear level in such a culture. Newly isolated tumor tissue was essentially haploid throughout, as was the tissue of origin, the gametophyte. However, the accumulation of nuclei at the duplicated haploid level ( 2 C ) of D N A marked the beginnings of endomitosis, the mechanism by which the polyploidy of these tumors had previously been shown to arise (Partanen et al., 1955). The old established lines of tumor had nuclear D N A levels which corresponded to the observed polyploidy, but also revealed that an appreciable part of the cell population was at a level other than that indicated by the chromosome counts. This indicated, therefore, that the polyploidy was secondary to the process of tumorization, and further, that the level of ploidy was quite variable within a single tumor. Also, superimposed upon the polyploidy was a variable aneuploidy. De Torok and White (1960), in a cytological analysis of primary explants of “normal and tumor wood’ of white spruce (Picea glauca), made extensive chromosome counts and found polyploidy and aneuploidy in both the normal and tumor explants, but noted a marked degree of difference between the two in these manifestations. The explants from the tumorous part of the tree were even more aberrant than those from the “normal” part. They emphasize that these were primary explants, and therefore the aberrancies are not explicable on the basis of the “abnormal” in vitvo conditions (a conclusion which they attribute incorrectly to Partanen, who concluded, in fact, that the observed nuclear changes were distinctive characteristics of tumors-cf. Partanen et al., 1955; Partanen, 1956, 1959). With respect to the spruce tumor cuItures, indeed they were primary explanis, but they were from old established t r ~ ~ o ~ s . Thus, perhaps a primary tumor of spruce would be more like the normal if the same relationship between nuclear abnormalcy and tumorization exists there as it does in many other cases, Unfortunately the etiology of the spruce tumor

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is unknown. Since the observed nuclear deviations by de Torok and White wer: in initial explants, and since no mechanism was noted which could explain their presence, the strong possibility exists that they are reflective of pre-existing co:iditions in oivo. The extent of deviation from the expected diploid number in the spruce “normal” tissue was quite remarkable (49%) of the nuclei counted were other than diploid), especially since these were in:tial explants. This would seem to exhibit considerable instability for “normal” tissue. In our experience with gymnosperm tissue in vilro, proliferating tissue obtained from embryos of Piiiur lambertiana, isolated and grown in separate culture, showed considerably more stability on both chemically defined and complex (coconut milk) media, over short and long periods of culture (Partanen, unpublished). Of 352 metaphases examined, 140 were at the diploid level and 12 were tetraploid. Thus, approximately 975% of the nuclei examined had the normal diploid chromosome number. There were no aneuploids apparent. A chromosome fragment was noted in only two figures, with no other aberrations evident. In a preliminary study of callus tissue obtained from embryos of Preudotsuga metzzzesii, essentially the same degree of chromosomal stability is evident, except that in this case all of the nuclei have been at the diploid level, again with no obvious aberrations (Partanen, unpublished). However, there are many possible factors to take into account in such differences as these. Certainly there may be species differences; spruce may be relatively unstable, Also, the source of and the method of obtaining the initial explant, as well as the subsequent culture methods may all be factors which may be reflected in the nuclear condition of the cells. In a later study, de Torok and Roderick (1962) examined the prevailing chromosome numbers in relation to growth rate in spruce tissue over a series of transfers in culture, and arrived at a positive correlation between chromosome number, rate of cell division, and rate of growth. They suggest that “there is a cause-and-effect relationship from chromosome number through rate of cell division to growth rate.” Among the very few successful tissue cultures of monocotyledonous plants that have been reported, and which included cytological observations, have been two on endosperm tissue. Straus (1954) successfully cultured the endosperm of corn, which has a basic triploid chromosome number ( 3 1 1 = 30). H e observed chromosome numbers that could be counted with some certainty up to 7-ploid with respect to the tissue of origin or 21-ploid with respect to the haploid number. Also prevalent were aneuploidy, bridges, fragments, lagging chromosomes, and “spindle disturbances.” Bridges were observed in about 307k of the figures. Haploidy was also noted. Norstog (1956) cultured the endosperm of rye grass, and although chromosome counts were not included, increased ploidy was observed. In this material, bridges were seen in about 50% of the figures. In the case of corn endosperm, the tissue of origin is known to

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undergo endomitosis in the course of its development ia vivu (Duncan and Ross, 1950; Punnett, 1953), and chromosome bridges are a regular phenomenon in certain instances (McClintock, 1939). Thus, the polyploidy and bridging observed in vitro might well be expected on the basis of the “normal” in vivo chromosomal behavior. An unusual tissue culture with respect to its origin is that obtained from the pollen of Gingko biloba by Tuledce (1957). The cells from which it arose were haploid, but in the process of proliferation in vitw it also developed some polyploidy, although there are no data to indicate the frequency or degree. The observations of Mitra et al. (1960) on carrot tissue in vitro are interesting in that they apparently show a difference in the response of the tissue with respect to the culture methods employed. Carrot tissue explants on solid medium showed the normal diploid chromosome number in all dividing nuclei, with no aberrancies noted. (However, it is not clear whether these observations extended beyond the initial explant). The same tissue, when grown in liquid culture as free cells and groups of cells, showed polyploidy to varying degrees in separate strains. Also noted were multinucleate cells, anaphase bridges, and one haploid figure. Haplopuppus gracilis is a much more suitable subject for cytological analysis, having a diploid chromosome number of 4. Cytological observations on Haplopappu-r tissue in vitw have been made by Reinert and Torrey (1961 ) and Mitra and Steward (1961). Reinert and Torrey showed that the medium can be a factor in determining the frequency of polyploidy. In the presence of coconut milk much more polyploidy was observed than on a synthetic medium. Their cultures were grown on solid media. Mitra and Steward encountered more nuclear aberrations in their studies on Hupl0papp.m cultures. However, since their numerous media contained coconut milk perhaps their observations are to that extent consistent with those of Reinert and Torrey. In addition to the normal diploid nuclei, Mitra and Steward found polyploidy, apparent somatic pairing, haploidy, pseudochiasmata, chromatid breaks, chromosome fragmentation, and multiple nuclei. The polyploidy was found to go as high as 16-ploid, although tetraploids were the most frequent. Another factor, or set of factors, to consider in comparing the results of Reinert and Torrey with those of Mitra and Steward involves the method of culture. The former used solid media whereas the latter used liquid media, with the vessel rotated at 1 r.p.m. (Blakely and Steward, 1961). As indicated by the studies on carrot (Mitra et al., 1960), this method of culture may be responsible for considerable nuclear aberrancy. Another favorable cytological tool which has recently been studied in vitro is viciu faba (Venketeswaran, 1962). This material was also found to manifest a number of aberrations, including polyploidy, aneuploidy, chromosomal bridges, and fragments. These studies were also done in liquid media, agitated on a shake table. Previous work with Vlcia has involved the study of induced

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divisions of mature tissues in vivo (Coleman, 1950) and D N A measurements in individual nuclei of normal and crown gall tissues (Rasch et al., 1959). Both of these studies showed that endopolyploidy is a common occurrence in mature tissues of the plant. In Pisum sativum tissues in culture, Torrey (1959) observed essentially the same types of nuclear deviations that have already been described, i.e., polyploidy, aneuploidy, bridges, fragments, rings, and binucleate cells. H e showed that the level of ploidy predominant among the dividing cells could be influenced by the medium, the prevalence of diploid cells being favored by a synthetic medium whereas a complex medium (yeast extract) favored the tetraploids. The same effect could be obtained by using kinetin or certain of its analogs added to the synthetic medium. Hence, it is possible to control the division of diploid or tetraploid cells in a mixed population by manipulating the medium. There was no evidence as to how the polyploids occurred in the cultures. Presumably there were nuclei of various levels of ploidy in the initial explants, Pisum having been demonstrated to manifest endopolyploidy (cf. D’Amato, 1952). However, on the basis of Torrey’s data, the polyploidy very probably did not all arise by endomitosis. The counts consistently showed levels of 2n, 4n, 8n, and 12n. On the basis of the 4n and 8n, one could assume that perhaps endomitosis did produce the polyploids. However, since endomitosis produces a geometric progression, the occurrence of the 12n nuclei suggests a more involved series of events. To arrive at 12n from a basic 2n calls for asynchrony somewhere in the replication process. There appear to be no good evidences of differential replication of entire chromosome sets in the same nucleus ; hence one must find a series of events that is more likely in terms of known mechanisms. This may involve a multinucleate cell, a result of the failure of cytokinesis, with subsequent asynchrony in the separate nuclei and later fusion of the separate nuclei. There are evidences of asynchrony in tissue culture cells (Straus, 1954; Mitra et al., 1960), showing nuclei within the same cell in different stages of the mitotic cycle. Torrey’s report included binucleate cells, and some of his photographs of “lobed nuclei” (1959, p. 210) are very reminiscent of nuclear fusion. (Some of our own observations on Viciu fuba in culture show a series of events from binucleate to fusion nuclei, see Fig. 2, D-F.) Perhaps on the basis of all the preceding reports of polyploidy in vitro, it might appear to be a safe generalization to state simply that plant tissues in culture can be expected to become polyploid. However, there is at least one report of a tissue which apparently does not show any inclination toward nuclear increases. Partanen (1959) analyzed the nuclear D N A levels in cultured tuber tissue of Helianthus tuberosus (Jerusalem artichoke) under a number of conditions and found that the nuclear D N A levels remained strictly within the diploid limits, as was also found to be the case in the tissue of origin. These findings were entirely consistent with the observations of Kupila (1958), who

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FIG, 2. Nuclei of plant tissue culture cells. A-F. Viriu faba. A. Normal diploid metaphase, x 600; B. Tetraploid metaphase, x 600; C . Anaphase bridge, x 600; D-F. Series showing binucleate cells with nuclear fusion to form lobed nucleus, x 800. G-I. Nicotiunu glutinoJa, x 1000. G. Normal root tip mitosis; H and I. Polyploid mitoses in tissue culture, note lagging chromosomes in I.

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found that the nuclei of crown gall of Helianthm amun.r (sunflower) itz z h o underwent no apparent change from the normal stem cells, which were uniformly diploid. Tschermak-Woess (1956) has pointed out that presence or absence of endopolyploidy appears to be a characteristic trait of closely related plants. A similar conclusion becomes apparent upon examining listings of its observed occurrence in various plants (cf. D’Amato, 1952). Hence, the process of endomitosis appears to have a genetic basis. Plant tissues in vitro are very probably capable of manifesting any or all of the natural in vivo tendencies of the cells involved, this manifestation being dependent upon the presence of conditions, chemical and/or physical, which will elicit such a response. Hence, perhaps the generalization that can be made is that if a certain condition such as somatic polyploidy is evident in v(vo, it can be expected to occur in vitro.

B. ANEUPLOIDY Thus, polyploidy in plant tissue cultures is explicable in terms of known mechanisms, either endomitosis or simply the failure of cytokinesis with subsequent nuclear fusion, or a combination of these. These mechanisms still maintain euploidy, however, since entire sets of chromosomes are involved. A frequent concomitant of changes in ploidy is aneuploidy, with chromosome numbers varying considerably (Straus, 1954; Partanen et al., 1955 ; Torrey, 1959; Mitra and Steward, 1961; de Torok and White, 1960; Venketeswaran, 1962). Accompanying phenomena often observed include some or all of such aberrations as chromosomal bridges, fragments, lagging chromosomes, and multipolar spindles. However, neither Partanen et al, (1955) nor de Torok and White (1960) saw any evidence of such occurrences. This would mean either that these phenomena did not occur, or if they did, the observations were not made at the time when they were prevalent. Where such disturbances are seen, an explanation for the aneuploidy is apparent. In their absence either no mechanism is put forth (de Torok and White, 1960) or the possibility of nondisjunction of chromosomes is suggested (Partanen et al., 1955) on the basis of other observations (e.g., Levan and Hauschka, 1953) as a potential mechanism. The occurrence of bridges and fragments is highly reminiscent of the effects of ionizing radiations (cf. Lea, 1946) or of various radiomimetic substances (Wilson, 1960). For example, the incorporation of structural analogs of naturally occurring purines produces many of the types of aberrations mentioned, including bridges, fragments, and “sticky chromosomes.” This is attributable to defects in the nucleic acid (Biesele, 1958) which gives rise to faulty chromosome structure and separation followed in some cases by fusions which lead to structural rearrangements. How such breaks can occur under the various in vitro culture conditions is not known. However, possibly several factors in the culture process can be contributing. An indication of the kind of factors that

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may be considered comes from the observations of Rieger and Michaelis (1958), who noted that the simple procedure of presoaking seeds of Vicia fabu for 72 hours would in itself produce a large number of breaks, 65Yo of the cells observed being aberrant. They attributed this effect to anaerobiosis during the soaking period. It has been shown that the rejoining of breaks induced by ionizing radiations is dependent upon respiration, more specifically upon the presence of adenosine triphosphate (ATP) (Wolff and Luippold, 1958). These observations would seem to be related, although the actual cause of the breaks in the soaked Vicia seeds remains obscure. Rieger and Michaelis refer to the unknown agent as an “automutagen,” i.e., some sort of endogenous factor. Keck and Hoffmann-Ostenhof (1952) showed that an extract of the seeds of Phaseolw vzrlgmis (kidney bean) would induce chromosomal fragmentation in Allizrm cepu (onion). Rutishauser (1956) suggests that in much of what is called spontaneous chromosome breakage a disturbance of the cell metabolism is suspect, as seen, for example, in certain hybrids. In the case of tissue cultures, in which the cells are in essentially a foreign environment, it is conceivable that some agent present could interfere with nucleic acid synthesis to the extent of causing similar breaks in the chromosomes. Further, an interference with the normal rate of respiratory activity could enhance the effect by reducing the supply of endogenous A D , thereby slowing down or reducing the natural repair of the breaks. Another factor which contributes to an increased rate of chromosome breaks is simply the age of the seeds (Jackson and Barber, 1958), the effects being essentially the same as those caused by ionizing radiations. Similar aberrations are noted in diff erentiated cells that are induced to divide by treatment with high concentrations of auxin (Therman, 1951). These evidences are suggestive that important considerations in interpreting the nuclear cytology of plant tissue cultures should be the nature of the tissue of origin with respect to such factors as age, prior physical events, physiological state in vivo, as well as physical factors in the process of obtaining an aseptic explant, e.g., heat, chemical treatment, etc. Further factors to be considered are chemical factors in the medium, the degree of aeration, the physical nature of the substrate (whether liquid or solid), and the presence or absence of agitatioc. All of these would seem potential factors, or at least should be suspect until shown to be otherwise.

C. HAPLOIDY Another kind of change in ploidy that is seen occasionally in plant tissue cultures is a reduction in chromosome number to the level of haploidy (Straus, 1954; Mitra et al., 1960; Mitra and Steward, 1961). Again there appears to be more than one way of arriving at the same effect. In a system in which aneuploidy is prevalent, conceivably gradual loss of chromosomes in some nuclei can eventually lead to the vicinity of the haploid number, although to arrive

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at such a condition and yet preserve one intact genome would seem to be of extremely low probability if the process which is producing the aneuploidy operates purely at random. Reductions of a more gross nature have been described occasionally. One type is the so-called “reductional grouping” of chromosomes in which mitotic chromosomes appear segregated into relatively distinct groups (cf. Huskins, 1947, 1948, 1952). In later stages of division, if this type of grouping persists and goes to an effective culmination it involves a multipolar spindle, such as are occasionally seen in plant tissue cultures (Straus, 1954). Spontaneously they are quite rare in occurrence, but by treatment of the material with nucleic acid salts their frequency can be increased (Kodani, 1948). The subsequent analysis of the phenomenon by Patau and Patil (1951) indicated that the total picture is a general effect upon the cell and that many of the observed cytological deviations can be explained on the basis of a disturbed spindle mechanism. Similar effects can be induced by prolonged low temperature treatment (Huskins and Cheng, 1950). Srinivasacher and Patau (1958) in a study of reductional groupings in onion root tips concluded that although somatic reduction by means of reductional groupings was likely, the probability of a mitosis being a reductional grouping which has a complete genome is approximately in the order of 10P. It is interesting to note that among those observing haploidy in tissue culture, several included substances in the medium which contained nucleic acid components (Straus, 1954; Mitra et al., 1960; Mitra and Steward, 1961; Venketeswaran, 1962). All made note of this possible relationship. The relatively low frequency of such reduced cells occurring in the tissue cultures can fall within the expected range if the large number of cells comprising the tissue culture is taken into consideration. Further, such cells once having arisen, can, conceivably multiply. The observations of de Torok and White (1960) indicated an appreciable number of cells which had less than the haploid number of chromosomes (hypohaploid). This is again a remarkable feature of their material, Piced glmca, since hypoploidy is generally considered a nonviable state. Some of their cells showed but three chromosomes, which would represent approximately one-quarter of the basic genome; that is, about three-quarters of the genome was missing. Yet, if the nucleus was in division, and that was the only nuclear material present in the cells, their observation raises the perplexing question concerning the necessity of the full genome. The fact that the nuclei were in division would suggest that the cells were still physiologically active. The alternatives that these observations leave are either that much of the genome of Picea glazlca is not essential for the (limited) functioning of the cell or that there is rather free exchange of materials between cells. Earlier studies in plants had indicated that the only way in which a hypohaploid could continue to survive was through having a relatively unimpeded access to the cytoplasm of an

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adjacent cell with a complete genome (Barber, 1941; Sax, 1942). An earlier reported case of apparent hypohaploidy in human uterine epithelium (Therman and Timonen, 1951) appears now to have been an artifact (Hsu, 1959). Another type of mechanism by which somatic chromosome reduction may occur appears to involve “somatic pairing” of chromosomes. Although observed regularly in certain insect tissues (Grell, 1946) this phenomenon is quite rare in general, and even more so in plants. However, the observations of Mitra and Steward (1961) on their cultures of Huplopupptls gracilis do suggest an association of homologous chromosomes. This behavior is then assumed to be correlated with the presence of haploidy. The course of events from the paired condition to one of haploidy seems uncertain. Chromosomal pairing, alone, does not automatically assure a reduced chromosome number. To accomplish a reduction in chromosome number, a second division without an intervening replication cycle is necessary. The events subsequent to chromosome pairing in Huplopdppus cultures would be an interesting process to follow, being one of the few cases of this type of apparent reduction in somatic cells. Hopefully, subsequent work will enable further studies and permit greater control and predictability of the process. It might be appropriate to suggest that another way of arriving at a haploid number of “paired” chromosomes would be to have two processes occurring simultaneously in a cell population. One would be a means of producing haploids, perhaps by reductional groupings. The other would be endomitosis. If a haploid cell, then, underwent one cycle of endomitosis, the resulting diplochromosomes might very much resemble “pairs of homologs” which in fact they are, though not “paired,” in the sense of having come together.

VI. Cytological Technique The foregoing discussions have mentioned a number of factors that can be contributing to the types of nuclear aberrations described in plant tissue cultures. These have indicated possible factors existing in the plant materials and in the various processes involved in culturing them. However, another area of consideration that should enter the total interpretation is that of the cytological techniques employed. Many of the plant tissues used in these studies are less than ideal cytological materials. Often they were not selected for their nuclear characteristics, the cytological observations being a secondary or incidental part of the total investigation. Consequently, in trying to deal with difficult materials, various facilitating devices or procedures are often employed. Caryology seems to have its share of erroneous observations. For example, many of the data on chromosomal variation in animal cells, both in uivo and in uitro, are now considered to be erroneous (Hsu, 1959). This has become evident through the application of

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improved techniques, the accurate determination of the human chromosome complement being a good case in point. Therefore, the interpretation of cytological observations in plant tissue cultures, and in plant materials in general, should include a consideration of the cytological techniques used. Thus, although the much more aberrant nuclear condition found in Haplopdppus by Mitra and Steward (1961) than by Reinert and Torrey (1961) may very possibly be due to differences in media or culture methods, there seems to be a notable difference in the cytological techniques as well. It seems unfortunate that most of the materials on which Mitra and Steward base their cytological observations had been treated with 8-hydroxyquinoline and a-bromonaphthalene prior to fixation. Treatment with 8-hydroxyquinoline is known to produce a wide range of cytological effects, at least in some materials, from the desired “c-mitosis” (for increased facility of chromosome counting) to radiomimetic effects, and a whole pattern of mitotic deviation termed a “d-mitosis” (Tjio and Levan, 1950, 1 9 5 4 ) . As was pointed out by Partanen el nl. (1955), when the concern is with the true cytological condition, analytical aids such as colchicine, paradichlorobenzene, and the like are to be avoided since they change the true relationships of chromosomes. For this very reason they are invaluable in routine chromosome counting, but by virtue of the nature of their action they are possible sources of artifact in analyses such as these, where the major point is chromosomal behavior. Other sources of error can arise as artifacts introduced by various seemingly inconsequential practices in the making of squash preparations. For example, some cytologists have the practice of tapping the coverslip in order to disperse the material further so as to produce a flatter preparation. This procedure can be demonstrated to produce such artifacts as broken chromosomes, unrealistic distribution of chromosomes, and loss of chromosomes by extrusion from apparently intact protoplasts. The latter is a particularly important point to consider when trying to establish the existence of aneuploidy.

VIJ. Morphogenetic Studies A. TISSUE CULTURES The foregoing observations on plant tissue cultures and cell cultures do of course constitute an end in themselves if the objective is to study nuclear behavior of cells grown apart from the influence of the intact organism. In that respect the plant tissue culture technique is a valuable cytological tool. Usually, however, the concern is not in cytology per se, but rather, in some meaningful relationship. One such frame of reference is morphogenesis, e.g., a consideration of the morphogenetic potentialities of cells which are being grown apart from the plant. This is the direction toward which the original concept of plant tissue culture was directed. On the basis of the preceding cytological

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discussion it is of interest to see what, if any, relationship can be seen between the nuclear condition of the cells involved and the degree or kind of morphogenetic expression that can be elicited. Ideally, to test the potentialities of any cell selected in vivo or in vZti.0 one would have but to remove that cell from its existing context, place it on a suitable substrate, and observe its subsequent behavior under a variety of controlled conditions. That, of course, is the ideal; in reality, at the present state of available techniques it can be approached, though not attained. There should always be the realization that the limiting factor may not be the cell’s potentiality, but rather the experimental conditions to which it is being subjected. Before the culture of single isolated cells became attainable in any manner, the most hopeful approach seemed to be to work with groups of cells, i.e., with excised portions of plants or with callus masses derived from such explants, and to study the effects of various factors or conditions on them. This may be at the level of histology, i.e., the expression of cellular organization in the callus (e.g., Gautheret, 1957). This type of organization may still be at a relatively rudimentary level. In the intact plant, the apical meristem is seen to play an important role in determining the morphogenetic pattern of the stem below it (cf. Wetmore and Wardlaw, 1951). Since a mass of callus is essentially without over-all organization, it presents an object for testing the morphogenetic influence of the apex. Wetmore and Sorokin (1955) grafted buds of Syringu vzllgaris (lilac) into callus obtained from cambial explants of the same plant. After a period of growth in vIIYo, interdigitation of tissues is evident between the scion and the host callus. The influence of the bud on the callus is evident in the differentiation of vascular strands in the previously parenchymatous callus. The work of Jacobs (1952) showed that auxin, produced by the young leaves and the apex, is a limiting factor in the differentiation of xylem in the plant body below. This same agent could be demonstrated to be a factor in the Syringa callus by making an incision, as for a graft, but by introducing auxin instead of the bud. The position of the resultant vascular differentiation was shown to be related to the concentration of the auxin introduced, the higher the concentration of the auxin, the further away from the incision the vascular strands differentiated. This demonstrated that the same morphogenetic influences can work in vitro as do in tfzio. A somewhat similar study by Clutter (1960) was done on excised tobacco pith cylinders maintained on a non-growth-promoting medium in v . h . The introduction of indoleacetic acid (IAA) through a glass pipette inserted into the pith cylinder over periods of 32 and 42 days caused cell divisions and differentiation of xylem cells in the pith tissue. This involves the induced in vitm differentiation of cells which were all laid down in vivo. These results are somewhat a t variance with those of Das et ul. (1956) who found that both

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auxin and kinetin (exogenous) were necessary to induce cell divisions in excised pith tissue of tobacco. However, the observations of Clutter were made over a longer period of time. Also, there may be differences between strains of tobacco in these responses (Skoog, 1957). Another approach has been the treatment of callus with various single agents or conditions in an attempt to produce and control the formation of organized structures. For example, certain tissues, notably tobacco callus, display a propensity toward the initiation of organization in the form of shoots and roots. By the manipulation of the medium with specific additives such as IAA and adenine (Miller and Skoog, 1953), or IAA and kinetin (Skoog, 1957), this phenomenon of organ regeneration can be regulated. Similar regeneration in tobacco callus was induced by submerging the callus in the medium instead of growing it on the surface (White, 1939). Reinert (1959) obtained shoot formation and small plantlets in a carrot tissue which previously had been producing only roots. The change was brought about by transferring the tissue from a complex medium (with coconut milk) to a synthetic one. These are but selected examples of the manner in which morphogenetic studies can be carried out with tissue explants and callus tissue in vitro. In general, they lack the kind of cytological observations which would be relevant to the present consideration. However, they do suggest experimental approaches into which such cytological observations as the nuclear condition of the tissue and the differentiating cells or regenerating structures, might profitably be incorporated. B. CELL CULTURES Attempts directed toward the culture of “single cells” have apparently evolved two concepts of the term. On the one hand, it seems to mean the culture of large numbers of cells in a liquid medium, the objective being to culture them as “single cells” rather than as groups or clusters. On the other hand, the concept of “single cell culture” can mean the culture of single cells alone, i.e., isolated from other cells. The latter would be the ideal morphogenetic tool. However, in either sense of the expression, the results to date still leave something to be desired. The culture of isolated cells away from other cells is most difficult to attain. The culture of single cells in numbers in liquid media has not, strictly speaking, been achieved (cf. Torrey and Reinert, 1961). But at least it can be approximated; such cultures generally consist of groups or clusters of cells as well as numbers of single cells. The inherent tendency of cells arising from the division of existing cells to remain attached to one another is the complicating factor. Initial approaches to this problem involved, for example, the use of oxalate to inhibit the formation of calcium pectate (Northcraft, 1951), or the occurrence of callus cultures which are more friable or “mushy” than usual (Reinert, 1956). Although the inoculum for a liquid culture may

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consist essentially of single cells, subsequent divisions will give rise to groups of cells. The single cells found in such cultures apparently arise through sloughing off from clusters. The degree of dissociation of cells can be controlled by the composition of the medium (Torrey and Reinert, 1961). This mass culture of dissociated cells, besides having become a tool in itself (e.g., Nickell, 1956; Steward et al., 1958b; Tulecke and Nickell, 1959; Torrey and Reinert, 1961), also lends itself to the culture of isolated cells by supplying such cells in an uninjured state (Muir et al., 1954, 1958). The present consideration will be concerned only with cytological and morphogenetic implications and applications of single cell cultures, in mass and isolated. The culture of isolated plant cells is generally hampered by the problem that a suitable substrate for the single cell of any angiosperm has not yet been defined. A fresh preparation of the same medium which supports the growth of a cell suspension or a callus of a specific strain is found to be inadequate for an isolated cell of that same strain. This problem has been circumvented in two ways. One has involved the “nurse” technique. Muir et al. (1954, 1958) nurtured cells isolated from liquid cultures by placing them on small pieces of filter paper which in turn are placed upon a callus tissue growing in uitto. The filter paper prevents direct contact between the isolated cell and the callus. In this manner isolated cells can be grown into a clonal callus mass which can eventually be grown independently on a suitable agar medium. This is a useful method for the establishment of clonal lines. However, it has a major drawback which prevents its application to the study of cellular differentiation and morphogenesis in that the isolated cells are at best difficult to observe. Another adaptation of the “nurse” tissue method which does permit microscopic observation of the isolated cell is that used by Torrey (1957). H e utilized a hanging drop of agar on a coverslip suspended over a well, with a nurse tissue centrally located and the isolated cells at the periphery. Only a limited amount of cell division was noted, however. Another approach to the same problem is that applied by Jones et al. (1960), who used a simple microculture chamber into which the isolated cell was placed along with some of the liquid medium in which it had been growing. This medium would already be “conditioned” for the growth of such cells. This method affords excellent optical conditions for observation of the cells in intimate detail. The initial cells were seen to undergo repeated divisions, to produce colonies of up to a hundred cells. Presumably such clusters of cells could be continued indefinitely in tissue culture if some provisions had been made either to replenish the medium or to transfer the group of cells, perhaps to a larger chamber of the same design, until it would survive the conventional tissue culture methods. This basic approach, however, seems to offer promise if it could be improved. Yet, these methods, i.e., the “nurse” technique and the “conditioned medium”

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microculture both fall short of the ideal situation since they do not utilize defined media. That is, there is no knowledge as to what the “nurse” tissue contributes to the cell nor what the constituency of the “conditioned” medium may be. Of course, with this knowledge, it might be possible to synthesize an adequate medium. Also, it may be that there are considerations other than simply the chemical composition of the medium, e.g., physical factors may also be important. Also, in the “nurse” technique the cells are not growing alone but rather in relation to other cells which are present, though not in contact. Another method of obtaining clones from cells which are in liquid culture is that of Bergmann (1960). The culture growing in liquid is passed through two filters of a suitable sited mesh which will allow only single cells or, at most, pairs of cells to pass through. These cells are then plated out in quantity onto an agar surface in a petri dish. The cells may then be observed at moderate magnifications. This technique does eliminate the necessity for both “nurse” cells and “conditioned” medium. However, it again deals with populations of cells, rather than truly isolated cells. From the point of view of a “clean” experiment in morphogenesis, there would be no substitute for isolated cells, alone, on a defined medium which is satisfactory for the growth and division of the cell. By utilizing a similar technique, we have established clonal lines of fern gametophyte tumors, some of which were isolated in 1958 and have been in continuous culture since then through repeated transfers (Partanen, unpublished). The original tumor is removed from its agar medium, partially dissociated by shaking briefly in a liquid medium, and a portion of the cell suspension removed with a fine pipette, to limit the size of pieces in the inocuIum. This sample is plated onto a simple agar medium in an optically Hat petri dish; single cells are located, marked, and photographed. The subsequent development is recorded photographically. The established clones of single cell origin are tumors just as the original tissue, and are once again as heterogeneous. For example, a single tumor of single cell origin has been found to have varying chromosome numbers among its cells (Partanen, unpublished). Again this, just as the Bergmann technique (1960), involves a population of separate cells on a single surface. An interesting application of the Muir technique for growing single cells into clones was made by Braun (1959) in testing the potentialities of cells comprising a teratomatous crown gall growth in Nicadana tabucum. The teratoma appears to be intermediate between normal and fully tumorous growth and is characterized by a continued ability to produce rudimentary shoots. An old established teratoma was placed into liquid culture and some of the resulting free cells were isolated and “nursed” on callus of Nicotiana glutimsa. Of a large number of isolates, a very few grew, all of which became characteristically teratomatous. Braun concluded that the cells of the teratoma were all “terato-

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matous,” i.e., that they did not represent a mixture of normal and tumorous cells. He then obtained a full recovery by repeated grafting of teratoma-produced shoots into the tops of normal plants. Eventually the grafted shoots developed to the flowering stage. Seeds were produced which germinated to form normal Nicotiaiza tahacum plants. On this evidence Braun concluded that somatic mutation at the gene level is an unlikely explanation for crown gall tumorization. Presumably the agent was something cytoplasmic which could be outgrown by the cells of the rapidly growing shoots. This experiment was a beautiful application of new techniques to an existing problem, i.e., a demonstration of the potentiality of a single cell of a teratoma when it is suitably nurtured. However, the conclusions of such an experiment should not be overextended; they say nothing about any other type of plant tumor, nor crown gall. Unfortunately the truly disorganized crown gall tumor cannot be tested in this same manner since (by definition) it does not produce any rudiments of organization. Indeed, the difference between teratoma and tumor may be reflective of a basic difference between either the nature, site, or degree of the primary causal lesion. Certainly, we are nowhere near the point of being able to generalize on the nature of the basic change in a cell that is causal to its tumorization. In some cases (e.g., Braun’s teratoma) the factor seems to be of a nonpermanent nature, probably cytoplasmic. On the other hand, in the case of radiation-induced fern gametophyte tumors, somatic mutation is the most attractive hypothesis (cf. Partanen, 1958; Partanen and Nelson, 1961). It would seem reasonable to expect that not all tumorous aberrations arise from the same type of basic cell change. Any morphogenetic manifestation, such as tumorization, could conceivably arise at the level of the gene, or through a dysfunction of any link in the chain of events from gene to ultimate expression. The work of Steward and his numerous associates (cf. Steward et al., 1961) is both an interesting and stimulating series of applications of plant tissue and cell cultures to problems of morphogenesis and cytology. The work with carrot (Steward, 1958; Steward et al., 1958a, b) involved placing several explants into a large rotating vessel containing liquid medium. In time there were cells and cell clusters in suspension ; these were transferred into fresh medium where they grew and multiplied. Some cell aggregates grew in size and developed roots. When placed on solid medium they also produced shoots and therefore developed into entire plants, some of which eventually flowered, displaying normal meiosis. The descriptions of the behavior of free cells in the liquid medium included several types of developmental patterns, i.e., cell proliferation from small isodiametric cells ; tubular filamentous growth ; internal divisions in existing large polynucleate cells ; and a process resembling “budding,” i.e., the formation of small papillae at the surface of the cell, which increase in size and get cytoplasm and a nucleus from the large cell. Histological examination of cell clusters presumably in the series of events leading to normal morphogenetic

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expression included organization reminiscent of embryonic stages of carrot. It was observed that although the cells in the suspension were aberrant in various ways, the normal plants or roots which arose from cell clusters were invariably normal diploids. This would suggest that possibly the genetic balance of the normal, intact genome is best suited to normal expression, i.e., that the various aberrants would be less likely to attain normal expression. A similar observation was made by Partanen et al. (1955) ; fern gametophyte tumors with prevailing aberrant nuclear divisions would occasionally give rise to normal prothallial growth, which was characterized by euploid chromosome numbers. The work of Steward et al. constitutes a very nice demonstration that at least some somatic cells of plants do retain totipotentiality. However, because of the manner in which the early stages arise in the liquid cultures, it is not possible to follow the developmental pattern from cell to organism. In fact, there is no evidence that such a continuity of events occurs. If, as Torrey and Reinert suggest (1961), the free cells arise by being sloughed off from larger pieces or clusters, the continuity from carrot tissue to regenerated carrot in Steward’s case may well be by way of the fragments and not ever involve the free cells. Perhaps an application of Bergmann’s (1960) filter technique could be used for limiting the size of pieces transferred. Further, perhaps an application of the pIating technique would permit observation of cells which would eventually give rise to organization. This might be expected since Reinert ( 1959) obtained essentially the same response from tissue cultures grown on solid media. However, the frequency of single cells which eventually give rise to complete plants may be so low as to reduce to impracticability the probability of having observed such a cell continuously in its development.

VIII. Conclusions Relatively little definitive work has been done in the general area of developmental plant cytology through the application of the tissue or cell culture techniques, Most observations reported are incidental to other considerations, or they simply report the behavior of the nucleus, offering little or no correlation with numerous factors that may be relevant. Yet, there would seem to be great potential in this approach. However, definitive work in this area calls for careful consideration of the numerous factors that may apply. For example, the interpretation must include a consideration of nuclear behavior in the plant and tissue of origin, both in meristematic and differentiated cells, for these are clearly reflected in vitro. Then, countless other factors attendant to the in vitvo technique must be considered, such as physical and chemical factors during the excision, sterilization, growth, and transfer of tissues or cells. Finally, since an objective of a cytological study is the behavior of the nucleus, the preparation of the material for visualization should be as straightforward as possible so as to minimize artifact.

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Observations of polyploidy and other nuclear deviations in plant tissues are largely explicable as a disorganized manifestation of normal tendencies of somatic cells (Partanen, 1959). Very probably cells in vitm do nothing other than what they may do somewhere in zri~o,but just as other cellular manifestations in tissue culture may be sporadic, so may the nuclear condition. The prevailing nuclear condition of a tissue culture can be seen to be responsive to differences in the medium (Torrey, 1959) and in the culture method (Mitra et a/., 1960). Thus, there are tools available for the experimental manipulation of these processes. In some cases, e.g., Huplopupp~s,the problem may be one of finding conditions which minimize the aberrations, thereby supplying as “dean” a point of departure as possible for the full utilization of this excellent cytological tool. Hopefully, future work will put greater emphasis on individual cells and their behavior. A promising approach would seem to be the microculture technique, with the necessary improvements, and perhaps coupled with periodic photographic records or with microcinematographic studies (e.g., Mahlberg, 1961) of the same cell over a long period of time. Such studies, with various environmental factors carefully controlled, could be invaluable to a correlation of various events with the eventual fate of a cell. That is, through an imaginative application of the tools and techniques now available, much could be learned about developmental plant cytology, and about cytological and developmental processes in general.

ACKNOWLEDGMENT The original work reported herein was supported in part by grants (Nos. CY-3335 and C-6251) from the National Institutes of Health, United States Public Health Service, and by Grant No. E-269 from the American Cancer Society.

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