CELL DIVISION IN RELATION TO CYTODIFFERENTIATION IN CULTURED PEA ROOT SEGMENTS

One mm-thick segments cut 10–11 mm proximal to the root tip of germinating seeds of garden pea Pisum sativum were cultured in sterile nutrient medium containing auxin in the presence and absence of kinetin. In the absence of added cytokinin, pericyclic proliferation occurred, the cortical tissues showed no proliferation and were sloughed off, and a callus tissue of diploid cells was formed. In the presence of kinetin concentrations from 0.1–1.0 ppm cortical cells of the segments were induced to divide, beginning at the third day. From experiments with ³H-thymidine incorporation at different times of culture, from cytological squash preparations and from histological sections it was shown that the cortical cells stimulated to divide by cytokinin underwent DNA synthesis prior to division, were polyploid, and following cell division rapidly underwent cytodifferentiation at 5–7 days to form mature tracheary elements. At 10 days, when over 300,000 new cells had been formed per segment about 16% of these cells had formed tracheary elements. It was concluded that cytokinin, together with auxin, was essential for the initiation of DNA synthesis in the cortical cells, for their subsequent division, and finally for their specific cytodifferentiation.     

Interaction of auxin, cytokinin, and gibberellin on cell division and xylem differentiation in cultured explants of Jerusalem artichoke.

Dark-cultured explants of parenchymatous cells isolated fromJerusalem artichoke tubers were induced to divide and differentiateas tracheary elements on Murashige and Skoog medium containingdifferent combinations of plant growth-hormones such as auxin(IAA), cytokinin (zeatin), and gibberellin (GA₃). Addition ofauxin to the growth-medium induced after a short lag period,very rapid cell division which was followed by differentiationof some of the divided cells as tracheary elements. At the optimallevel of IAA (5.0 mg/liter), the percentage of tracheids differentiatedwith respect to the total number of cell population was 13.54.When the explants were cultured in the presence of both auxin(IAA 5.0 mg/liter) and one cytokinin (zeatin 0.1 mg/liter),not only a strong interaction on cell division and trachearyelement formation was observed but also an increase in the percentageof tracheids differentiated in relation to the total cell population.Auxin-gibberellin and auxin-gibberellin-cytokinin treatmentsalso produced interaction on cell division and cytodifferentiation.In explants treated with the three growth-hormones about 20%of the total cell population differentiated as tracheary elements.Further, all the hormonal treatments gave different patternsof cytodifferentiation which reflected meristematic patterns. ¹ This research was supported by a grant from C. N. R. Italy. (Received April 18, 1973; )     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

Direct Evidence for Cytodifferentiation to Tracheary Elements without Intervening Mitosis in a Culture of Single Cells Isolated from the Mesophyll of Zinnia elegans

A serial observation of the process of tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird provided the first direct evidence for the cytodifferentiation without intervening mitosis. Percentage of the tracheary elements formed without cell division was about 60% of total tracheary elements formed on the 4th day of culture. The number of tracheary elements formed without intervening mitosis was not reduced in the presence of colchicine at the concentrations blocking cell division. These facts clearly indicate that cell division is not a prerequisite for tracheary element differentiation in this system.     

Hormonal Control of Cell Proliferation and Xylem Differentiation in Cultured Tissues of Glycine max var. Biloxi

The relationship between tracheary element differentiation, cell proliferation and growth hormones was examined in agar-grown soybean callus. The time course of cell division and tracheary element formation in tissues grown on a medium containing 5 x 10(-7)m kinetin and 10(-5)m NAA was determined by means of maceration technique. After a slight lag period, a logarithmic increase in cell number was observed through the twelfth day of the culture period. Cell numbers increased at a considerably slower rate after the twelfth day. The rate of tracheary element formation varied with the rate of cell proliferation. Tracheary elements increased logarithmically during the log phase of growth. As the rate of cell division decreased after the twelfth day of culture, the rate of tracheary element formation also decreased. In the presence of 10(-5)m NAA, cell number increased as the kinetin concentration was increased between 10(-9) and 10(-6)m. However, tracheary element formation was not initiated unless the kinetin concentration was 5 x 10(-8)m or above. When the Biloxi callus was subcultured repeatedly on media containing 10(-8)m kinetin, a tracheary element-free population of cells was obtained. This undifferentiated tissue produced tracheary elements upon transfer to a medium containing 5 x 10(-7)m kinetin. In the presence of 5 x 10(-7)m kinetin, NAA stimulated cell proliferation between 10(-7) and 10(-5)m, but no tracheary elements were formed without auxin, or with 10(-7)m NAA. Neither NAA nor kinetin at any concentration tested stimulated tracheary element formation in the absence of an effective level of the other hormone. However, 2,4-D at 10(-7) or 10(-6)m promoted both cell proliferation and tracheary element differentiation in the absence of an exogenous cytokinin.     

Establishment of an Experimental System for the Study of Tracheary Element Differentiation from Single Cells Isolated from the Mesophyll of Zinnia elegans

Single cells were isolated mechanically from the mesophyll of adult plants and of seedlings of Zinnia elegans L. cv. Canary bird. When single cells isolated from the first leaves of seedlings were cultured in a liquid medium in the dark with rotation, they differentiated to tracheary elements with a reasonable degree of synchrony in the 24-hour period between days 2 and 3 after culture. The proportion of tracheary elements as a percentage of total cells reached nearly 30% 3 days after culture. Factors favoring cytodifferentiation were certain optimum levels of both α-naphthalene-acetic acid (0.1 milligram per liter) and benzyladenine (1 milligram per liter), a low concentration of ammonium chloride (0 to 1 millimolar), and an initial cell population density in the range 0.4 to 3.8 × 10⁵ cells/ml. It was possible to follow analytically the sequence of cytodifferentiation in individual cells in this system.     

HORMONE-INDUCED ENDOREDUPLICATION PRIOR TO MITOSIS IN CULTURED PEA ROOT CORTEX CELLS

One-mm-thick cortical explants excised aseptically from 10-11 mm behind the tip of 3-day-old roots of the garden pea, Pisum sativum, cv. ‘Little Marvel’ were cultured on a synthetic nutrient medium supplemented with auxin or auxin and cytokinin. Nuclear DNA contents were measured in cells of the explants at the outset and at specified times during culture up to seven days. Fixed and sectioned preparations were stained with the Feulgen method using the DNA-specific dye auramin-O. Fluorescent microspectro-photometric measurements of individual nuclei were made from each cortical population. At day zero all cortical nuclei measured were either 2c or 4c with respect to their DNA content. In the presence of the auxins, indoleacetic acid and 2,4-dichlorophenoxyacetic acid, and the cytokinin, kinetin, DNA values increased to multiples of the 2c level with populations at the 8c and 16c level predominating after three days of culture as well as at seven days. In the presence of auxins alone no change in DNA values was observed during three days. Kinetin concentrations as low as 0.01 ppm were already effective. The data are interpreted to show that cytokinin, in the presence of auxin, induces two rounds of DNA synthesis prior to the first mitoses, the first round being connected with chromosome doubling by endoreduplication and the second one with normal mitosis. From this we inferred that tetraploid cells in leguminous root nodules might have arisen in the same way, i.e., by endoreduplication prior to the first mitoses induced by the rhizobial division stimulus, unless the chromosome number of root cortical cells had already been doubled by endoreduplication in the normally differentiating root systems.     

Tracheary Element Formation from Single Isolated Cells in Culture

Friable callus tissue of Centaurea cyanus L. was grown on a solidified synthetic nutrient medium (EBM-1) to produce a tissue with a low frequency of differentiated tracheary elements. Tissues were then suspended in liquid nutrient medium with agitation to produce a suspension which was filtered and the single-cell suspension resulting was used as inoculum for either cell suspension cultures or for plating of cells into solidified medium in Petri plates. Media for the suspension cultures were selected to favor cytodifferentiation of tracheary elements. Differentiated tracheary elements formed as early as 10 days and numbers of tracheary elements increased with time roughly in relation to the increase in total cell number. From plating experiments it was shown conclusively that single isolated parenchyma cells differentiated directly into single isolated tracheary elements, although this event was rare. More usual was the division of isolated cells to form small colonies and then the differentiation of one, several or all of the cells into tracheary elements. Comparisons are made between results with cell plating experiments and cell suspension cultures. Optimism is expressed for finding a cell suspension culture system for studying cytodifferentiation.     

Relationship between tracheary element differentiation and the cell cycle in single cells isolated from the mesophyll of Zinnia elegans

A relationship between tracheary element differentiation and the cell cycle was studied in single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird. Almost all nuclei of isolated mesophyll cells were at the 2 C level of DNA, indicating that almost all cells were initially in the G₁ phase and that somatic polyploidy was absent. Cultured cells underwent partially synchronous DNA replication at 42 h and mitosis at 54 h of culture, and the first cell cycle time was approximately 58 h.
The occurrence and timing of DNA replication and mitosis during cytodifferentiation to tracheary elements were investigated using microspectrophotometry, microfluorometry, tritiated thymidine autoradiography, and serial observation. More than 55% of the nuclei of the immature tracheary elements were at the 2 C level of DNA and were not labeled by continuous feeding with tritiated thymidine, providing clear evidence that these cells differentiated without interventing DNA replication. Some tracheary elements (approximately 30%) were formed after one round of the cell cycle, and others (less than 5%) were formed after passing through the S phase, but without intervening mitosis. All types of tracheary elements appeared simultaneously after 58 h of culture, and their patterns of increase in number were similar. From the results, we propose a hypothesis concerning the relationship between cytodifferentiation and the cell cycle.     

Is DNA synthesis required for the induction of differentiation in quiescent root cortical parenchyma?

Cortical parenchyma of pea roots normally does not divide nor differentiate as tracheary elements. After excision from the root these cells can be induced to undergo cell division followed by differentiation of tracheary elements in the presence of cytokinin. 5-Fluorodeoxyuridine prevents cell replication and cytodifferentiation. The thymidine analog, 5-bromodeoxyuridine, specifically prevents cytodifferentiation with little or no effect on the rate of cell replication. Thymidine can also prevent cytodifferentiation with little effect on cell replication. Thymidine reverses the effect of 5-bromodeoxyuridine (BrdU) or 5-fluorodeoxyuridine (FdU) when given simultaneous with or subsequent to either BrdU or FdU. BrdU given with FdU allows up to one round of cell replication but no cytodifferentiation. Differentiation is rarely observed unless the entire cell population has undergone the equivalent of two cell generations. These results are combined with those of other investigators to present an hypothesis that implicates DNA replication but not cytokinesis in the regulation of certain types of differentiation.     

Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary elementdifferentiation in lettuce pith explants was about ten timesgreater than the optimal concentration for callus proliferation.Related to this, the mean volume per tracheary element increasedwith increasing IAA concentration, 18-fold between 0.001 mMand 0.3 mM IAA. At the highest concentrations, some pith cellsappeared to differentiate directly into tracheary elements,without cell division, resulting in especially large trachearyelements. Tracheary strands developed at intermediate concentrationsof IAA, and led to a small increase in the mean length/breadthratio of tracheary elements. For tracheary elements differentiating from stem cambial derivatives,a reassessment of previous studies indicates that increase inauxin concentration brings greater tracheary element size atconcentrations up to the 0.03 mM optimum. Above this optimum,however, further increase in auxin concentration brings progressivelysmaller tracheary elements, as the high auxin curtails enlargementof the differentiating cells. This contrasts with the pith explants,in which tracheary element size increases with IAA concentrationmost markedly above the optimum concentration. The interpretationof these relations requires an understanding of the effectsof auxin concentration on interacting quantities such as initialsize of cells, rate of enlargement, and rate of differentiation. Lactuca sativa, lettuce, IAA concentration, pith explants, tracheary element dimensions     

Pattern of cell division and wound vessel member differentiation in coleus pith explants

When pith parenchyma explants are taken from Coleus blumei plants and cultured on an agar medium containing sucrose and indoleacetic acid wound vessel members differentiate in 10 days. The time course of wound xylem appearance and an auxin requirement suggest that this uncomplicated system is responding in a manner comparable to wounded Coleus plants and cultured stem segments.

Histological examination and cell size comparisons confirm that parenchyma cells divide before differentiating. When colchicine is used to prevent mitosis no tracheary elements differentiate. Following the time course of this cytodifferentiation histologically shows that xylem differentiates from cells that are the products of several cell divisions.

     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : III. Tubular Gland Cell Cytodifferentiation

Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types—some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.     

Hormone regulation of development in plant cells

Summary Hormonal stimulation of dedifferentiation and redifferentiation can be studied in explanted, cultured plant tissues. Some of the questions about development which one would like to answer with such a system revolve around the role of quiescence in the stabilization of the differentiated state, the role of replication in the stimulation of redifferentiation and the means by which cells are brought out of the quiescent state. Such a system also offers the potential for revealing the level(s) at which plant hormones operate in the stimulation of replication and differentiation since the responses to hormones can be achieved in vitro. The pea-root cortical parenchyma system has been utilized as a model system in the study of cytokinin plus auxin stimulation of redevelopment of mature, quiescent root cells. The first detected response of the root parenchyma to excision and culture with both of these hormones is an enhanced rate of RNA synthesis between 9 and 12 hr after the initiation of culture. DNA synthesis is stimulated 36 to 39 hr after RNA synthesis (after 48 hr in culture). During this 48-hr period various cytological changes have been observed which are compatible with renewed nucleic acid synthesis, but cytological changes have not been observed prior to the onset of hormone-stimulated RNA synthesis. The first mitoses and cytokineses occur after 60 and 72 hr, respectively. Terminally differentiated tracheary elements are first formed in these cultures after 120 to 168 hr when both the cytokinin and auxin are present at adequate levels. Studies employing inhibitors suggest that tracheary element differentiation is dependent upon the DNA replication that normally accompanies cell replication. Temperature probes of the period between the initiation of cultures and the appearance of the terminally differentiated tracheary elements have been initiated and, in conjunction with previous studies employing inhibitors and analogues, may allow one to distinguish between a variety of potential models of hormone-stimulated redifferentiation. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. Supported in part by grants from the National Science Foundation (GB 36948) and the Public Health Service (RR 07092).     

Inhibition of xylogenesis by morphactin in pith parenchyma explants of Lactuca

Pith parenchyma explants of Romaine lettuce (Lactuca salivaLinn. var. Roman?) incubated in the dark for 7 days at 25?Con a nutrient medium containing sucrose, IAA. and kinetin exhibitedextensive differentiation of tracheary elements. The additionof CFL to the medium strongly inhibited tracheary element formation.The lack of tracheary strand formation in the CFL-treated explantssuggests the inhibition of auxin transport. Conclusive evidencethat CFL influences the anatomy of differentiating xylem elementswas lacking. The addition of CFL to various combinations ofxylogenic media was not stimulatory to xylem element formationbeyond the differentiation response observed in the absenceof CFL. Unique patterns of tracheary element formation producedby cytokinin media containing IAA, 2,4-D, and NAA, respectively,were abolished by CFL. As indicated by counts of total trachearyelements formed per explant, the addition of cysteine to a CFL-containingmedium reversed the inhibitory effect of CFL. Tracheary strandformation was not re-established in the explants cultured onthe cysteine+CFL medium. Tracheary element formation was completelysuppressed by TIBA. Cysteine had a slight effect on the inhibitionof differentiation by TIBA. These observations suggest thatCFL inhibits some sulfhydryl- containing system involved eitherin the process of xylem differentiation or in some prerequisiterole necessary for the induction of tracheary element formation. (Received December 27, 1972; )     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Exogenous Methionine as a Nutrient Supplement for the Induction of Xylogenesis in Lettuce Pith Explants

The induction of xylogenesis in explants of lettuce pith parenchymawas greatly influenced by the presence of exogenous methionine(0·025 to 0·05 µM) in the culture medium.At the various concentrations of methionine tested, trachearyelement differentiation was stimulated in the majority of theexplants. Differentiation, however, was markedly depressed ina small number of explants grown under the same cultural conditionsin the presence of methionine. Cytodifferentiation in controlexplants, cultured on a similar medium lacking methionine, gaveconsistent tracheary cell counts with little variation. Thesedata are consistent with the hypothesis that theenhanced productionof ethylene, due to the presence of methionine as a substrate,plays a role in the initiation of xylem differentiation. Theconcentration of ethylene in the cultured tissue may be a criticalfactor in determining whether the hormone will stimulate orsuppress the initiation of cytodifferentiation. Some thick-walledand pitted cells were observed, and these may represent partially-differentiatedxylem elements.     

Rapid differentiation of tracheary elements in cultured explants of Jerusalem artichoke

the culture of Jerusalem artichoke (Helianthus tuberosus L.) tuber explants on filter paper discs moistened with liquid medium resulted in rapid and consistent xylem differentiation. The number of tracheary elements increased in discrete steps, the first at 48 h with a second at 56–58 h, following partially synchronous mitoses at 20 and 30 h. Factors favouring xylem cell differentiation were optimum levels of both an auxin and a cytokinin, low medium nitrogen concentrations, small volumes of medium, and high culture temperatures. A cell counting method employing Feulgen-stained nuclei and suitable for quantifyings small numbers of immature tracheary elements is described.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - BAP benzylaminopurine - GA₃ gibberellic acid     

Characteristics of trophoblast cell reproduction in the connective zone of the rat placenta. II. Determination of the degree of ploidy of mitotic shapes

Morphological and cytophotometric studies have been made on polyploidization of placenta connective zone cells. Measurement of the DNA content in mitotic figures show that within a period of development ranging from day 13 to day 14 the bulk of mitoses (up to 25%) become tetraploid and octaploid. This may suggest that polyploidization of placenta connective zone cells proceeds via incomplete polyploidizing mitoses. Among tetraploid and octaploid mitotic figures, there are those corresponding to all the mitotic stages, from prophase to telophase. Consequently, mitosis in tetraploid and octaploid cells can reach telophase. In such cases polyploidization is likely to follow the acytokinetic mitotic pattern. A question of a certain maximum level of polyploidy that may be reached by cells due to the incomplete mitosis is discussed.     

Patterns of Tracheary Differentiation in Lettuce Pith Explants: Positional Control and Temperature Effects

When lettuce pith explants were cultured for 14 d on a xylogenicmedium, tracheary elements differentiated in greatest numbersbetween 25 and 30 °C. Numbers were depressed at lower temperaturesby slower development and at higher temperatures by adverseprocesses. The data did not support previous suggestions ofa great stimulation of xylogenesis above 30 °C and of aspecial sensitivity to low temperatures. Tracheary elements differentiated in various spatial patterns:as clumps in the peripheral callus, as strands which extendedradially and longitudinally from some of these clumps, as individuallarge tracheids especially at the more extreme temperatures,and as short strands associated with nodules and roots thatformed at favourable temperatures. We suggest that indoleacetic acid (IAA) has various roles inthe positional control of these tracheary patterns: (1) IAAdestruction at the explant surface leads to concentration gradientsthat inhibit tracheary induction close to the surface; (2) IAAtransport from the source in the culture medium to sinks especiallyat the explant surface, coupled with autocatalytic flow facilitation,leads to canalization along pathways that become meristematicand then trachcary strands; (3) the IAA flux (and associatedproton flux) along these pathways tend to orient cortical microtubulesat right angles to the flow, by some mechanism as yet unknown,and hence to control the orientation of tracheary element elongationand secondary wall banding. These suggestions, supported bymorphometric studies of tracheary element dimensions and orientations,and by experiments with localized IAA application, lead to ageneral interpretation of the development of polarity in plants. IAA, Lactuca sativa, lettuce, pith explants, positional control, temperature effects, tracheary element differentiation