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.     

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.     

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; )     

DNA synthesis, cell division and specific cytodifferentiation in cultured pea root cortical explants

Pea root segments cut 10–11 mm behind the tip of germinating seedlings were prepared by removal of the central cylinders with a tissue punch. These cortical explants were cultured aseptically on nutrient medium containing auxin with and without added cytokinin. In the absence of kinetin, the cortical cells enlarged and separated but failed to show DNA synthesis, mitosis, cell division or subsequent cytodifferentiation. In the presence of 1 ppm kinetin, cortical nuclei showed ³H-thymidine incorporation beginning between 24 and 32 hr; mitoses began about 48 hr, reaching a maximum of 6% at 60 hr. From an initial number of 8000 cells per segment, the cell count increased to 37,000 by day 7 and 140,000 by day 21. At the outset all mitoses were tetraploid; with time the proportion of tetraploid mitotic cells decreased and an octaploid population increased. A frequency of less than 10% diploid mitoses was observed after day 5. Only 25% of the cortical cells showed initial labeling. Beginning on day 7 tracheary elements differentiated from cortical derivatives. By day 14 about 25% and by day 21 about 35% of the total cell population had formed tracheary elements. As a system for analysis in biochemical and cytological terms, pea cortical explants represent an excellent system for the study of cytodifferentiation.     

Tracheary element differentiation is correlated with inhibition of cell expansion in xylogenic mesophyll suspension cultures.

To test the hypothesis that xylogenesis is coupled to cell growth suppression, cell expansion in Zinnia elegans L. var. Envy mesophyll suspension cultures was manipulated by varying the extracellular osmolarity and the effect on xylogenesis was examined. Cell expansion and tracheary element differentiation were inversely related along a gradient of extracellular osmolarity ranging from 200 to 400 mOsm, supporting the hypothesis that tracheary element differentiation is coupled to cessation of cell expansion. Above 300 mOsm, reduction in the number of cells that differentiated into tracheary elements coincided with an increase in the number of plasmolyzed cells as extracellular osmolarity was increased, indicating that plasmolysis inhibits tracheary element differentiation, although not specifically. Using the plasmolysis method we showed that cellular osmolarity within populations of isolated Zinnia mesophyll cells ranges from 250 to 600 mOsm with a mean of 425 mOsm. The broad range in cellular osmolarity within Zinnia mesophyll cell populations, coupled with inhibition of differentiation in the low range due to cell expansion and in the high range due to plasmolysis, may help explain why tracheary element differentiation in Zinnia suspension cultures is never complete nor perfectly synchronous and enable further optimization of this culture system.     

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.     

Autolysis during in vitro tracheary element differentiation: formation and location of the perforation

Tracheary elements differentiated from isolated Zinnia: mesophyll cells were observed at various times of culture under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends in single tracheary elements. In double tracheary elements, which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results suggest not only that a single tracheary element has its own program to form a perforation at one end without being affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.     

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.     

In vitro studies on xylogenesis in citrus fruit vesicles. II. Effect of pH of the nutrient medium on the induction of cytodifferentiation

The initial pH of the nutrient medium influenced the type of cytodifferentiation occurring in cultured isolated fruit vesicles of Citrus limon (L) Burmann var. Assam lemon. Neither callus formation nor cytodifferentiation was found at pH values below 3.0. Three types of cytodifferentiation were found after 30 days culture in the presence of a liquid Murashige and Skoog (MS) basal medium supplemented with MS vitamins, indoleacetic acid (IAA) (10 mg/l), kinetin (0.2 mg/l), and sucrose (3% w/v): sclereids, xylem fibers and tracheary elements. The greatest numbers of sclereids and tracheary elements were found in callus grown on a medium with an initial pH 5.0–6.0, whereas pH 7.0 was optimal for the formation of xylem fibers.     

Morphogenesis in cell colonies grown from Convolvulus cell suspensions plated on synthetic media

Filtered cell suspensions of cultured callus tissue derived from the roots of Convolvulus arvensis L. were plated out on synthetic agar nutrient media in petri plates. Cell colonies which formed from the single cells or small cell groups in the suspension showed a considerable range of developmental patterns depending upon the physical and chemical environment to which they were exposed. Variation of the auxin and kinin concentrations and the nature and concentration of the source of reduced N compounds had the most profound effects on colony development. High auxin favored cell enlargement, high kinin favored the development of compact colonies composed of many small cells. Both auxin and kinin were required for cell colony formation. Cell differentiation responses which were observed but not subject to experimental control included formation of starch- and crystal-storing cells, differentiation of tracheary elements, formation of cellular filaments, and development of chlorophyllous tissue. Organ initiation was studied in cell colonies developed directly from plated cell suspensions and in cell colonies subcultured on various nutrient media. Bud initiation was produced repeatedly on media containing NAA at 10^-8 to 10^-6 m combined with kinetin at 10^-6 m . Root initiation was induced infrequently and unpredictably. Once roots had been formed from cell colonies derived from cell suspensions, the roots could be subcultured and induced to form buds; these in turn grew into whole plants. Subculture of young cell colonies to media containing different combinations of growth substances made possible a study of the effects of auxin and kinin on organization of primordia by the cell colonies. By following marked single cells plated on synthetic media, it was possible to produce single-cell clones which under proper nutrient conditions were induced to form buds. The value of the combined techniques of cell suspension culture and cell plating for the study of the physical and chemical factors influencing cell differentiation and organized development are pointed out.     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements

As the first step toward understanding the involvement of endogenous brassinosteroids (BRs) in cytodifferentiation, we analyzed biosynthetic activities of BRs in zinnia (Zinnia elegans L. cv Canary Bird) cells differentiating into tracheary elements. The results of feeding experiments suggested that both the early and late C6-oxidation pathways occur during tracheary element differentiation. Gas chromatography-mass spectrometry analysis revealed that five BRs, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol, and 6-deoxoteasterone, actually existed in cultured zinnia cells and culture medium. Quantification of endogenous BRs in each stage of tracheary element differentiation by gas chromatography-mass spectrometry exhibited that they increased dramatically prior to the morphogenesis, which was consistent with the idea that BRs are necessary for the initiation of the final stage of tracheary element differentiation. Moreover, the proportion of each BR in culture medium was quite different from that in cells, suggesting that specific BRs are selectively secreted into medium and may function outside the cells.     

A Secreted Factor Inducs Cell Expansion and Formation of Metaxylem-Like Tracheary Elements in Xylogenic Suspension Cultures of Zinnia

Conditioned medium from mesophyll cell-suspension cultures of Zinnia elegans L. has striking effects on cell expansion and tracheary element differentiation when applied to cultures of freshly isolated mesophyll cells. These effects include (a) induction of early cell expansion, (b) delay in differentiation by 48 h or more, (c) reduction in the synchrony of differentiation, and (d) early formation of very large, metaxylem-like tracheary elements. Like reduced osmotic potential and buffering at pH 5.5, conditioned medium appears to have its primary effect on cell expansion. Partial characterization of the expansion-inducing factor indicates that it is heat stable, of low molecular mass, and is resistant to protease. It also binds reversibly to concanavalin A but is not adsorbed by charcoal. We suggest that the secreted factor may be an oligosaccharide involved in the coordination of cell expansion and differentiation and the regulation of the protoxylem-like to metaxylem-like transition in xylogenic suspension cultures.     

Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose

A procedure is described for the reproducible establishment of rice cell suspension cultures from callus of embryo origin. Protoplasts were readily isolated from cell suspension of four rice cultivars, including japonica and indica types, when maintained in an amino acid-based culture medium. Sustained protoplast division from two japonica genotypes has been obtained in agarose solidified culture medium. An increase in the agarose concentration from 0.6% to 1.2% (w/v) produced a marked improvement in protoplast survival, division and plating efficiency. Protoplast division and plating efficiency frequencies of up to 26% and 0.5%, respectively, were obtained at the higher agarose level. The protoplast-derived calli were similar in appearance to explant-derived morphogenic callus and produced distinct embryo-like structures.     

In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata)

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 μM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.     

Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions

Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 mum brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.     

REGENERATION OF LIRIODENDRON TULIPIFERA (FAMILY MAGNOLIACEAE) FROM PROTOPLAST CULTURE

Protoplasts were isolated enzymatically from suspension cultures of two embryogenic lines of yellow-poplar (Liriodendron tulipifera L.) and cultured in droplets of a regeneration medium supplemented with 1 mg/1 2,4-D and 0.25 mg/1 6BA and solidified with agarose. Suspension cultures grown in the light yielded substantial numbers of protoplasts, while insignificant numbers were isolated from cultures grown in the dark. The protoplasts reformed cell walls within three days, and 75 percent of them divided at least once within one week in culture. Embryogenic suspension or callus cultures were regenerated by placing agarose droplets containing protoplast-derived microcalli directly into liquid conditioning medium or onto plates of conditioning medium solidified with agar. Embryogenesis was obtained by transferring the callus to a hormone-free induction medium.     

Transition of G1 to early S phase may be required for zinnia mesophyll cells to trans-differentiate to tracheary elements

We have used the Zinnia elegans mesophyll cell system, in which single isolated leaf mesophyll cells can be induced to trans-differentiate into tracheary elements in vitro, to study the relationship between the cell division cycle and cell differentiation. Almost all cells go through several rounds of division before characteristic features of tracheary element formation are observed. The addition of aphidicolin, a DNA synthesis inhibitor, blocks cell division but not cell differentiation in the zinnia system. Low concentrations of aphidicolin, which possibly delay cells in the early S phase, can significantly enhance levels of tracheary element formation. In contrast, roscovitine, an inhibitor of cyclin-dependent kinase activity, decelerates the cell division cycle and inhibits tracheary element formation with similar dose responses. Cells blocked in S phase and then transferred to roscovitine-containing medium can divide once, indicating that roscovitine may target the G1/S transition, but do not differentiate. Cells inhibited in G1/S in roscovitine-containing medium that are subsequently blocked in S phase by transfer to aphidicolin-containing medium, do not divide but do differentiate. Taken together, our results indicate that cells may be required to transit the G1/S checkpoint and enter early S phase to acquire competence to trans-differentiate to tracheary elements.     

Effect of high salinity on tracheary element differentiation in light-grown callus of Mesembryanthemum crystallinum

This study investigated the inhibitory effects of NaCl on tracheary element (TE) differentiation in light-grown callus of ice plant Mesembryanthemum crystallinum L., a halophyte which adaptes well to saline environments. When ice plant callus was grown in a modified Linsmaier-Bednar and Skoog culture medium containing no NaCl (control medium), up to 20% of ice plant cells differentiated into tracheary elements during in vitro culture. Close examination of callus tissues stained with potassium permanganate revealed that tracheary elements were aggregated as discrete nodules. Some strikingly elongated tracheary elements were found in the macerated tissues. Experimental results indicated that adding 200 mM NaCl to the control medium reversibly inhibited the formation of tracheary element in the halophytic cells. The rate of tracheary element formation increased accordingly as the rate of cell growth in control medium. In the presence of high salt, the degree of tracheary element differentation remained low through the growth cycle. The inhibitory effect of salt on tracheary element differentiation was overcome by adding 10 mg l⁻¹ salicylic acid, a known signaling compound that induces a diverse group of defense-related genes, including genes involved in reinforcing the host cell wall. Furthermore, microscopic examination revealed that most tracheary elements formed under this treatment (200 mM NaCl plus 10 mg l⁻¹ salicylic acid) were round shaped. The results suggest that high salt inhibits both the biosynthesis of secondary wall components and cell elongation ice plant in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tip growth in xylogenic suspension cultures ofZinnia elegans L.: Implications for the relationship between cell shape and secondary-cell-wall pattern in tracheary elements

Summary The relationship between cell expansion, cortical microtubule orientation, and patterned secondary-cell-wall deposition was investigated in xylogenic cell suspension cultures ofZinnia elegans L. The direction of cell expansion in these cultures is pH dependent; cells elongate at pH 5.5–6.0, but expand isodiametrically at pH 6.5–7.0. Contrary to our expectations, indirect immunofluorescence revealed that cortical microtubules are oriented parallel to the long axis in elongating cells. Pulse labeling of the walls of isolated cells with the fluorochrome Tinopal LPW demonstrated that xylogenic Zinnia mesophyll cells elongate by tip growth in culture. These results confirm that cortical microtubules in developing tracheary elements reorient before bundling to form transverse cortical microtubule bands. This rearrangement may allow the secondary cell wall pattern to conform to cell shape, independent of the direction in which the cell was expanding prior to reorientation.Abbreviations CMT cortical microtubules - Mes 2-[N-morpholino]ethanesulfonic acid - TE tracheary element