Differentiation of Vascular Tissues

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 alpha-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 x 10(5) cells/ml. It was possible to follow analytically the sequence of cytodifferentiation in individual cells in this system.

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

The Physiology of in Vitro Asexual Embryogenesis

Most classical plant hormones are also produced by pathogenic and symbiotic fungi. The way in which these molecules favour the invasion of plant tissues and the development of fungi inside plant tissues is still largely unknown. In this review, we examine the different roles of such hormone production by pathogenic fungi. Converging evidence suggests that these fungal-derived molecules have potentially two modes of action: (i) they may perturb plant processes, either positively or negatively, to favour invasion and nutrient uptake; and (ii) they may also act as signals for the fungi themselves to engage appropriate developmental and physiological processes adapted to their environment. Indirect evidence suggests that abscisic acid, gibberellic acid and ethylene produced by fungi participate in pathogenicity. There is now evidence that auxin and cytokinins could be positive regulators required for virulence. Further research should establish whether or not fungal-derived hormones act like other fungal effectors.

Plant hormones: a fungal point of view

The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. The terminal 0- to 1-millimeter root tip contained 43 to 44 times more free cytokinin on a fresh weight or a per cell basis than the next 1- to 5-millimeter root segment. Extracts of more proximal segments behind the tip contained no measurable free cytokinin. Acid hydrolysates of transfer RNA exhibited reproducible cytokinin activity. Bioassays revealed that the predominant amounts of free cytokinin and that present in transfer RNA were restricted to the extreme root tip. There was approximately 27 times more free cytokinin than the amount detected in transfer RNA in root apices.

Cytokinins in seedling roots of pea.

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 × 10−7m kinetin and 10−5m 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 10−5m NAA, cell number increased as the kinetin concentration was increased between 10−9 and 10−6m. However, tracheary element formation was not initiated unless the kinetin concentration was 5 × 10−8m or above. When the Biloxi callus was subcultured repeatedly on media containing 10−8m kinetin, a tracheary element-free population of cells was obtained. This undifferentiated tissue produced tracheary elements upon transfer to a medium containing 5 × 10−7m kinetin. In the presence of 5 × 10−7m kinetin, NAA stimulated cell proliferation between 10−7 and 10−5m, but no tracheary elements were formed without auxin, or with 10−7m 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 10−6m promoted both cell proliferation and tracheary element differentiation in the absence of an exogenous cytokinin.

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

The relationship between DNA synthesis and wound xylem differentiation was investigated in cultured stem segments of Coleus blumei. The addition of 50 micrograms of indoleacetic acid per liter to the culture medium resulted in a 400 to 500% increase in the number of wound vessel members formed in 7 days. However, the time course of wound vessel member formation was similar in segments cultured in the presence and absence of auxin. In either case, no wound vessel members appeared before the 3rd day of culture, while the majority of wound vessel members appeared on the 4th and 5th days of culture. 3H-Thymidine incorporation into DNA was used to measure changes in the DNA synthetic activity of the tissues during the culture period. Comparatively little 3H-thymidine incorporation occurred during the 1st day of culture. Maximum 3H-thymidine incorporation was observed on the 2nd day of culture, 2 days before the peak period of xylem differentiation. The rate of incorporation of 3H-thymidine into DNA decreased with increasing time in culture after the 2nd day. Auxin at 50 micrograms per liter had no effect on the time course of 3H-thymidine incorporation, although somewhat more 3H-thymidine was incorporated into DNA throughout the culture period in the presence of auxin. The magnitude of this effect was small when compared to the effect of auxin on xylem differentiation. The antimetabolite 5-fluorodeoxyuridine was shown to block DNA synthesis in the cultured stem segments. When the tissues were isolated on media containing 10−6m 5-fluorodeoxyuridine, wound vessel member differentiation was inhibited by approximately 80%, in both the presence and absence of auxin. Thymidine at 10−5m completely overcame the 5-fluorodeoxyuridine inhibition of wound vessel member formation. 5-Fluorodeoxyuridine was effective in blocking xylogenesis only when this substance was supplied to the tissues during the early part of the culture period. 5-Fluorodeoxyuridine had no effect on xylem differentiation when it was applied after the 3rd day of culture.

D. E. Fosket

Papers

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

The time course of xylem differentiation and its relation to deoxyribonucleic Acid synthesis in cultured coleus stem segments.