Tracheary elements that resemble secondary xylem in calli derived from the conifers, Torreya nucifera and Cryptomeria japonica
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Differentiated cells were recognized in calli derived from needles of Torreya nucifera and in calli derived from immature zygotic embryos of Cryptomeria japonica. Some differentiated cells resembled tracheary elements of primary xylem with spiral or reticulate thickening of cell walls. Other cells resembled tracheary elements with thick cell walls and bordered pits, which are features of secondary xylem. These tracheary elements were formed in cell clusters. Tracheary elements in calli of T. nucifera formed more highly developed structures, such as bordered pits and spiral thickening, than those of C. japonica. Cultured cells derived from conifers might provide a good model for studies of the differentiation of secondary xylem in vitro.
KeywordsBordered pits Conifers Secondary xylem Secondary wall Tracheary elements
Tracheary elements, such as tracheids and vessel elements, play an important role in water transport in plants and, thus, the formation of tracheary elements is an important aspect of xylem differentiation. Since wood consists of secondary xylem cells derived from the cambium of trees, a full understanding of the mechanism of differentiation of secondary xylem, and, in particular, of the deposition of secondary walls is needed for improvement of wood quality.
An in vitro model of tracheary element differentiation would be useful for investigations of the differentiation of secondary xylem. A model system, using isolated mesophyll cells of Zinnia elegans, was developed by Fukuda and Komamine  for studies of xylem differentiation and has provided extensive information at the cellular level . In addition, a system of tracheary element differentiation in vitro, using Arabidopsis thaliana cells in suspension, has been used for analyses of the cytoskeleton and related proteins during the formation of the cell walls [3, 4]. In such differentiation systems in vitro, the main products are tracheary elements that resemble primary xylem, with spiral or reticulate thickening of secondary walls. They do not yield tracheary elements that resemble secondary xylem, with broad areas of secondary wall thickenings and bordered pits. By contrast, an in vitro system for tracheary element differentiation using the conifer, Pinus radiata, developed by Möller et al. [5, 6, 7], yielded tracheary elements with helical, scalariform, reticulate and pitted types of secondary wall thickening. Therefore, we postulated that it might be possible to induce the formation of more highly developed types of tracheary element from cultured cells of conifers.
In this report, we describe the presence of tracheary elements in cultured cells of the conifers Torreya nucifera and Cryptomeria japonica. Some of these tracheary elements resembled secondary xylem, with broad areas of secondary wall thickenings and bordered pits.
Materials and methods
Cell culture in vitro
Calli were induced from young needles of Torreya nucifera trees that were growing in the campus of the Tokyo University of Agriculture and Technology, Fuchu, Tokyo. For induction of calli, young needles were placed on a modified version of MS medium , with the concentration of KNO3 changed to 3.4 g/l and without NH4NO3 but including 3 % sucrose and 100 mg/l myo-inositol, with the pH adjusted to 5.7, and solidified with 0.3 % gellan gum. As a plant regulator, 2,4-dichlorophenoxy acetic acid (2,4-D) were added to the medium at 1.99 mg/l. The needles and resultant calli were incubated at 25 °C in darkness, with subculture in the same medium at 4-week intervals.
Calli of Cryptomeria japonica were induced from immature zygotic embryos in a modified version of CD medium  in which the concentration of NH4NO3 was reduced to 800 mg/l, and to which 2,400 mg/l l-glutamine, 3 % sucrose, and 1 g/l myo-inositol were added. The pH was adjusted to 5.7, the medium was solidified with 0.2 % gellan gum , and 2,4-D was added at 0.66 mg/l as a plant growth regulator. The embryos and resultant calli were incubated at 25 °C in darkness, with subculture in the same medium at 4-week intervals.
Induction of the differentiation of tracheary elements
Calli of T. nucifera were transferred to suspension culture in liquid medium of the same composition as the solid medium but without gellan gum. Then cells were agitated on a rotary shaker at 110 rpm at 25 °C in darkness. Suspension-cultured cells were washed five times on nylon mesh with hormone-free subculture medium. Approximately 0.8 g (fresh weight) of cells was transferred to 20 ml aliquots of induction medium. The composition of the induction medium was the same as that of the hormone-free subculture medium but with the addition of activated charcoal at 5 g/l.
In the case of C. japonica, small pieces of calli, 4 weeks after subculture, were transferred to induction medium, which had the same composition as the hormone-free subculture medium, with the addition of activated charcoal at 5 g/l. All cultures were maintained at 25 °C in darkness.
Microscopic observations of tracheary elements
Cells on the upper side of calli that were harvested suspended in distilled water and observed by bright-field or polarized-light microscopy or differential interference contrast microscopy (Axioskop; Carl Zeiss, Oberkochen, Germany). We defined cells with strong birefringence as tracheary elements because of the presence of secondary walls with well-ordered cellulose microfibrils. The area percentage of tracheary elements was determined by calculating the ratio of total area of tracheary elements per total area of cells with image-analysis software (Image-J; National Institutes of Health, MD, USA).
Calli were stained for 30 min with a 0.01 % aqueous solution of safranin for observations of secondary cell walls and examined by confocal laser scanning microscopy (LSM310; Carl Zeiss) with a 590-nm long-pass filter and excitation by an argon ion laser (488 nm) [11, 12].
Tracheary elements in calli of Torreya nucifera
Tracheary elements in calli of Cryptomeria japonica
Induction of the differentiation of tracheary elements
In the case of C. japonica, when the calli themselves had included small numbers of tracheary elements (Fig. 3c), incubation in hormone-free medium plus activated charcoal increased the numbers of tracheary elements in calli (Fig. 3d). Image analysis showed that the area percentage of tracheary elements was 1.2 % in Fig. 3c and 31.0 % in Fig. 3d. By contrast, when calli already included many tracheary elements, activated charcoal had no obvious effect on the number of tracheary elements. The induced tracheary elements tended to be located in cell clusters (Fig. 3b, d) and almost all of them resemble primary xylem cells, having reticulate thickenings of their secondary walls.
We found tracheary elements in calli derived both from needles of Torreya nucifera and from immature zygotic embryos of Cryptomeria japonica. Some of the tracheary elements in calli of T. nucifera formed bordered pits that are characteristic of secondary xylem. Möller et al. [5, 7] also observed the development of pitted tracheary elements in calli of Pinus radiata. In our study, we found more highly developed pits and, even, bordered pits pairs (Fig. 2b, c). In addition, the tracheary elements in calli of T. nucifera had the spiral thickening of secondary walls that is a typical feature of tracheids of T. nucifera. Therefore, cultured cells of T. nucifera appear to provide a good model for the induction of the secondary xylem type of tracheary elements in vitro. By contrast, calli of C. japonica contained only tracheary elements with secondary wall thickenings typical of primary xylem, such as the helical or reticulate type. Thus, the nature of types of tracheary elements in calli appears to differ among species of conifers.
The numbers of tracheary elements in calli of T. nucifera and C. japonica increased after calli were transferred to hormone-free medium supplemented with activated charcoal. Möller et al. [5, 7] also observed such an increase in tracheary elements in calli of P. radiata in the presence of activated charcoal. Activated charcoal might absorb phenolic compounds and plant hormones from the incubation medium . A putative decrease in levels of plant hormones, such as auxin, in the medium might induce differentiation into tracheary elements. However, the induced tracheary elements had only the spiral and reticulate types of secondary wall thickenings (Fig. 6). Therefore, activated charcoal might be an effective enhancer of the number of tracheary elements in calli of the conifers T. nucifera and C. japonica but is not associated with induction of the development of tracheary elements.
Tracheary elements were frequently observed in cell cluster, and it seems plausible that the cells that differentiated first into tracheary elements might induce neighboring cells to develop into tracheary elements. Motose et al. [14, 15] identified the arabinogalactan protein “xylogen” as an inducer or promoter of the differentiation of tracheary elements in Zinnia system. Xylogen or some similar proteins might be involved in intercellular communication during the differentiation of tracheary elements in calli of T. nucifera and C. japonica.
In conclusion, we found that calli of T. nucifera developed tracheary elements that resembled secondary xylem with broad areas of secondary wall thickening and bordered pits. Such a system might be useful for studies of the mechanism of differentiation of secondary xylem cells in vitro.
This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (nos. 19580183, 2012009, 21380107, 22·00104, 23380105, 24380090 and 24·2976).