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Cell Biology International (2009) 33, 524–533 (Printed in Great Britain)
Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation
Peter Twumasiabc*, Jan H.N. Schela, Wim van Ieperenb, Ernst Wolteringd, Olaf Van Kootenb and Anne Mie C. Emonsa
aLaboratory of Plant Cell Biology, Department of Plant Sciences, Wageningen University and Research centre, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
bHorticultural Production Chains Group, Department of Plant Sciences, Wageningen University and Research Centre, Marijkeweg 22, 6709 PG Wageningen, The Netherlands
cDepartment of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
dAgrotechnology and Food Innovation, Wageningen University and Research Centre, Wageningen, The Netherlands


Abstract

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1mg/l each of benzylaminopurine (BA) and α-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.


Keywords: Cellulose, In vitro culture, Programmed cell death, Apoptosis, Tracheary element, Xylogenesis, Zinnia elegans.

*Corresponding author at: Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), PMB, Kumasi, Ghana. Tel.: +233 245 131806; fax: +233 516 4338.


1 Introduction

Xylem vessels and tracheids are important structures in higher plants due to their water conducting abilities and mechanical support (Tyree, 2003). The xylem cells originate from the root and shoot procambium during the early developmental stages of the plant and also from the vascular cambium during the secondary growth periods of the plant. For over a century, extensive work has been done to unravel the complex mechanisms involved in xylem formation and its hydraulic function in the plant (Aloni, 1987; Chaffey, 1999; Dengler, 2001). In just over two decades, our knowledge about xylogenesis both at cellular and molecular levels has increased more than ever before (McCann et al., 2001). For instance, there is a great deal of studies involving xylem formation focusing on understanding the mechanism of cellulose synthesis (Haigler et al., 2001; Mellerowicz et al., 2001; Cano-Delgado et al., 2003). The availability of xylogenic cell culture systems, such as Zinnia, Arabidopsis and Populus, has provided essential tools for an in-depth understanding of the xylogenesis process (Ye, 2002). Programmed cell death (PCD) is essential during formation of certain functional structures, in both animals and plants. It is also involved in defense mechanisms as demonstrated in the hypersensitive response (Iakimova et al., 2008). In animals this type of self-induced cell death is referred to as apoptosis, a term relating to the apoptotic bodies (or membrane-bound structures) resulting from the breakdown of the membrane at the end of the death process (Yang et al., 1999; Ranganath and Nagashree, 2001; Sanmartin et al., 2005). In animals, these apoptotic bodies are later engulfed through phagocytotic activity in the organism. Such apoptotic bodies have, however, not been found in plant cells during programmed cell death. Because of the involvement of the PCD in TE differentiation in the xylem (Fukuda, 1996; Roberts and McCann, 2000), more attention is being focused on the use of this simpler xylogenic system to study regulation of PCD in plants.

The in vitro xylogenic cell culture is suitable for the study of xylem development and differentiation studies due to the easy accessibility for manipulation, microscopic analyses and production of one simple cell type isolated from the complexity of the tissue. On the other hand, the whole plant xylogenic process is preserved. Since many molecular studies require extraction of ample amounts of the molecule under study, a highly efficient and synchronous TE differentiating cell culture would be necessary. The Zinnia elegans xylogenic cell culture, although introduced long ago (Fukuda and Komamine, 1980), continues to show the highest efficiency and synchrony in TE differentiation better than any of the recently introduced xylogenic cultures (Arabidopsis: (Oda et al., 2005); Populus: (Ohlsson et al., 2006)). At the time of Zinnia xylogenic cell culture discovery, the yield of TE was just around 30% (Fukuda and Komamine, 1980). More recently, there have been records of TE differentiation as high as 60% (Church, 1993; Fukuda, 1996).

Despite these achievements, different laboratories report of low and varying TE yields formation in the xylogenic Zinnia cultures (Gabaldon et al., 2005; Tokunaga et al., 2005; Oda and Hasezawa, 2006). The inconsistencies in the protocol for establishing the Zinnia culture, ranging from plant material to phytohormonal induction, may account for these differences. The aim of this work therefore was to produce a standardized and reproducible protocol for establishing higher yields of TEs in Z. elegans in vitro cultures. Also, modified cytological and biochemical methods were to be designed for monitoring progress of TE differentiation.

2 Results

2.1 Sequence of events during TE differentiation

Mechanically isolated Zinnia mesophyll cells have definite shapes, usually asymmetrically cylindrical and measuring 20–60μm in length. We have established in this work that phytohormone application 24–48h after cell isolation produces TE differentiation (24-h induction, 74% TE; 48-h induction, 76% TE). Also, a reliable viability measurement was achieved within this period. Viability of cells in the NICM observed over 5 days showed no significant changes, but the change was significant in the ICM (Fig. 2). Cells in the ICM maintained higher cell division as compared to those in NICM (Fig. 3). However, cells from ICM were smaller as compared with those from NICM (Fig. 4). Expansion of cells in the ICM occurred within the first 24h of the culture while in the NICM the expansion continued beyond 48h. In the ICM, this stage was immediately followed by an active secondary cellulose deposition on the cell wall leading to formation of cellulose bands.


Fig. 1

Time course for TE differentiation in Zinnia elegans suspension culture. The differentiation and lignification processes require 96h to complete. (Bar=20μm).


Fig. 2

Changes in the viability of Zinnia elegans mesophyll cells in culture during tracheary element differentiation. Concentration of inductive hormones (NAA and BA) were set at 1mg/l in the cultures. [ICM, inductive culture medium; NICM, non-inductive culture medium (control); N=100 cells and TEs; bars=SEM].


Fig. 3

Rate of cell division in suspension cultures of Zinnia elegans upon differentiation (white bars) and in control (black bars).


Fig. 4

Effect of tracheary element differentiation on the size of Zinnia mesophyll cells. Different letters indicate significant differences. (NICM, non-inductive culture medium, ICM, inductive culture medium; N=100; bars=SEM).





TE differentiation becomes visible 48h after treatment. This stage is characterized by sequential secondary cellulose cell wall deposition, nuclear condensation, vacuole rapture and DNA laddering. Finally, autolysis of the differentiating cells revealed various secondary cellulose band patterns. Various types of tracheary elements present in planta were recovered in the in vitro culture (Fig. 5). Lignification occurred later around 120h in the inductive cultures, about 12h from complete autolysis of the TEs. The sequence of events that occurred during TE differentiation in the Zinnia culture is schematically summarized in Fig. 6.


Fig. 5

Comparison of different types of tracheary elements in the Zinnia in vitro culture and those in planta showing different thickening patterns (annular, spiral, reticulate and pitted).


Fig. 6

Summary of events that occur during tracheary element differentiation in Zinnia elegans suspension culture. The entire process lasts for 96h from time of induction.



2.2 Vital staining with FDA

We observed that high levels of dead mesophyll cells in starting Zinnia culture either inhibited TE differentiation completely or significantly reduced TE differentiation. Moreover, since mature TEs are dead and hollow cells with secondary cellulose thickenings, the TE differentiation itself eventually results in lowering of the initial viability. Cell viability is therefore a measure of the progress of TE differentiation. Differentiating Zinnia cultures with initial cell viability of 60% or higher (Fig. 7) was found to produce workable amounts of TEs.


Fig. 7

Zinnia mesophyll cells stained with fluorescein-diacetate (FDA) for the calculation of cell viability. Viable cells are those emitting green fluorescence at 510nm, while the dead cells remain dark.


2.3 Nuclear condensation, DNA labelling with TUNEL and gel electrophoresis

At 48h of the culture, the nuclei of cells in ICM were condensed –losing the regular oval or round shape which is visible with DAPI staining. The nuclei of the differentiating cells disappeared between 72 and 96h period of the culture, a stage associated with extensive autolysis of the differentiating cells. These phenomenal changes were however not present in control cultures (Fig. 8I, J).


Fig. 8

Micrographs showing morphological changes in differentiating cells of Zinnia elegans in inductive culture medium. DIC image of freshly isolated mesophyll cells (A); DIC image of fully differentiated TE 96h after induction (B); autofluorescence of lignin in differentiated TE at 96h (C) – excitation=420nm; Calcofluor white stained cellulose of cells at 0-h, 72 and 96h of induction (D, E, F) - excitation=365nm, emission=410nm; FDA stain (0.005%) showing intact vacuoles at 48h (G), and collapsed vacuole at 72h (H) - excitation=488nm, emission 510nm; Nuclear staining with DAPI (1mg/ml) at 0-h (I) and 48h (J) after induction.(Bar=15μm).


The TUNEL positive nuclei (having DNA ladders or fragments) were observed in the differentiating cultures 36h after treatment, and the level increased linearly till 72h (Fig. 9a and b). The laddering in DNA occurred 12h earlier than nuclear condensation observed. TUNEL was negative in the control cultures at 72h. However, on agarose gel the DNA laddering was clearly visible at 72h in the differentiating treated cultures and negative in the control (Fig. 10).


Fig. 9

a: DNA fragmentation in nuclei as detected by the TUNEL assay in Zinnia elegans mesophyll cells during TE differentiation – control at 72h (upper left), ICM at 36 (upper right), 48 (lower left) and 72h (lower right). The fragmentation of the DNA in the nuclei is detected by the TUNEL assay visible as greenish fluorescence at 525nm emission wavelength. (Bar=30μm). b: Changes in nuclear DNA laddering (measured as TUNEL positive nuclei) during tracheary element differentiation in Zinnia culture. Control culture (without TE differentiation) was measured at 72h.


Fig. 10

Gel electrophoresis showing DNA fragmentation in Zinnia cell undergoing TE differentiation. DNA fragmentation is pronounced at 78h. The control (C) lane was obtained from non-inductive culture medium at 78h of culture establishment.



2.4 Lignin synthesis

Lignin is bluish-white under UV. The lignin was observed 96h or longer only in the ICM at a time when the TE differentiation had been completed (Fig. 8C). The undifferentiated cells in both ICM and NICM do not form lignin.

2.5 Secondary cellulose deposition and pattern formation

Staining of the cell wall with Calcofluor white revealed a steady increase in cellulose deposition from 36h to 72h in the ICM (Fig. 8D, E, F). Different wall patternings of the secondary cellulose deposition including annular, spiral, reticulate, pitted and blends of two or more were revealed after cellular autolysis. These were absent in the control cultures in which the cells could develop only the primary cellulose wall.

2.6 Vacuolar collapse

FDA staining showed clear distinction between the cytoplasm and the vacuole in TE differentiating cells. During autolysis when the tonoplast collapses, the cytosol and vacuoles become stained with FDA. At this stage, the differentiating and non-differentiating cells are easily recognized (Fig. 8G, H and Fig. 11).


Fig. 11

Changes in the level of cell populations with collapsed vacuoles upon induction of TE differentiation. Statistically significant differences are indicated by different letters. (Bars=SEM.).


3 Discussion

Xylogenic cultures of Z. elegans established from leaf mesophyll cells have high tracheary element (TE) differentiation (76%), and at the moment leads all the available xylogenic cultures including that from Arabidopsis thaliana (Oda et al., 2005). The associated high frequency and synchrony in TE differentiation of the Zinnia system, measuring over 76%, makes it useful especially for molecular studies involving isolation of ample amounts of molecular markers. Additionally, the Zinnia culture is suitable for studying mechanism involved in autonomous programmed cell death in plants, especially in the development of the vascular system. Another interesting feature of the Zinnia system is its high reproducibility in TE differentiation and the relatively short time necessary for the differentiation process. Nevertheless, the critical steps involved in setting up the culture must be carefully adhered to to maximize the potentials of the system. Also, the monitoring techniques applied to the TE differentiation process are fast and reliable, and therefore allowing the processes involved to be scrutinized.

Preconditioning of the isolated cells 24–48h after cell isolation was essential for successful induction of TE differentiation. However, a long preconditioning time did not promote higher TE differentiation. This could be due to exhaustion of nutrients in the un-refreshed culture and the associated low cell division occurring in the NICM. Such a culture limitation in the Zinnia system necessitates establishment of cultures from freshly isolated mesophyll cells. In this study, cell populations were found to increase significantly with the addition of BA (1mg/l) and NAA (1mg/l). However, this period of cell division is short (about 48h) as most of the cells later differentiate into TEs.

Wounding of cells initiate signal molecules that help to differentiate native cells into TEs. This might explain why old or sub-cultured cells that have healed do not respond to the differentiation induction. Thus, it is difficult, if not impossible, to maintain a mother culture that can be sub-cultured over a longer period of time and still maintain same level of TE differentiation. This means that every experiment will require preparation of freshly isolated mesophyll cells to initiate the xylogenic culture. TE differentiation in fresh Zinnia cultures is associated with production of wound or stress signaling molecules. Others have shown in vivo that wounds inflicted on vascular bundle cells cause local accumulation of signal molecules such as auxin to initiate vascular regeneration through transdifferentiation of localized parenchyma cells. These cells form new tracheary or vessel elements and sieve elements which connect ends of the severed bundle (Aloni, 1992; Nishitani et al., 2002).

Development of techniques that would allow in vitro induction of wound signals or stress inducing compounds such as reactive oxygen species (ROS) and exogenous cytokinin in the old or sub-cultured cells, might help in keeping the Zinnia cultures over a longer period and still maintaining adequate levels of TE differentiation in the subculture or older culture.

Apoptosis, a term commonly used for programmed cell death (PCD) in animals (Yan et al., 2006), is attracting much attention from many laboratories as a result of the recent surge in researchers aiming at retarding aging (Li et al., 2006). The TE differentiation process also recruits PCD in building a functional cell remains required for water uptake in plants (Groover and Jones, 1999). This is shown by TUNEL staining and gel electrophoresis. The Zinnia system is therefore a useful plant model for studying programmed cell death.

It has been shown in this work that, with the right conditions and techniques as indicated in this paper, the xylogenic Zinnia culture can be improved by conditioning the culture. With this approach, percentage TE as high as 76% percent was achieved. It is therefore recommend that Z. elegans var Envy be used as model plant for cellulose, xylogenesis, programmed cell death and other molecular research. This would also require whole genome sequencing.

4 Experimental procedures

4.1 Plant material

60 seeds of the Z. elegans var Envy – a cultivar commonly used for cell culture work – (Muller Bloemzaden BV, Lisse, Netherlands), were germinated in 30×40×7cm trays filled with peat-based commercial potting compost (Lentse Potgrond nr. 4; 85% peat, 15% clay, Lentse Potgrond, Lent, The Netherlands) in a greenhouse of the Wageningen University and Research, Wageningen, The Netherlands. 16-h day light of 200μmolm−2s−1 at 25°C, 8-h darkness at 20°C and a relative humidity of 70% were maintained throughout the growth period. Regular watering applied carefully at the base of the plants was necessary to prevent microbial contamination of the leaves from the soil. This precaution was found to be effective in preventing infection in the final cell suspension produced.

4.2 Cell isolation and culture

Isolation of mesophyll cells from leaves of the Z. elegans is a critical step as it influences the TE differentiation in the induced culture. An initial culture of mesophyll cells with cell viability of 60% or more was maintained throughout the experiment. We established that cultures that have initial cell viability below 40% either did not differentiate at all or differentiated with very low TEs (data not shown). Also, healthy mesophyll cells ensured high level of TE formation. The following protocol was therefore developed for isolation of cells with high percentage differentiation.

A schematic representation of the various steps involved in the culture is also shown in Fig. 2. Except for the first two steps (1 and 2), all the steps were carried out in a sterile airflow cabinet with benches sterilized with 70% ethanol. Sterile hand gloves were worn throughout the cell isolation and culture transfers.

1.

30 sets of first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were harvested by cutting the stalks connecting the leaves to stems. The right stage for the seedlings was when the first true leaves have just fully expanded and the primordium of the second set of leaves has just been initiated (Fig. 1). This stage is critical because older leaves tend to produce lower rates of TEs.

2.

The leaves were surface sterilized in a 500ml beaker containing 300ml of cold 0.15% NaOCl and 0.001% Triton X-100 solution for 10min. Sterilization process was immediately stopped whenever dark-green spots were observed on the leaf surfaces even before the 10min to prevent necrosis of cells by the hypochloride solution.

3.

The leaves were rinsed three times in sterile Milli-Q water.

4.

Gentle mechanical maceration of the leaves in 30ml cold non-inductive culture medium (NICM; without phytohormones) was accomplished in a mortar. Although a higher application of mechanical force would result in higher yield of mesophyll cells from the leaves, reduction in the cell viability associated with it would result in lower %TE. Typically, Zinnia mesophyll cells are loosely attached to the epidermal layers and therefore require a slight force to release into surrounding CM.

5.

The mixture was then filtered through a sterile 50μm nylon mesh, and the filtrate containing the mesophyll cells was collected into a sterile 50ml beaker.

6.

The filtrate was transferred to a 10ml centrifuge tube and spun to pellet the mesophyll cells at 200× g or 1100rpm for 1min.

7.

The supernatant was carefully removed and discarded. The pellet made up of the mesophyll cells was washed 3× with NICM.

8.

The cleaned mesophyll cells were resuspended in 10ml NICM and 200μl of it transferred to a fresh Eppendorf tube for viability and cell density measurements.

The mesophyll cell suspension were cultured in 3ml volumes in a 6ml sterile culture plates either before or after treatments with phytohormones or other drugs. Averagely, 30 leaf pasirs produce 300ml mesophyll cell suspension at 105 cells/ml cell density.

4.3 Zinnia culture medium

The basic nutrient composition of the Zinnia culture medium (CM) is shown in Table 1. The composition is closely related to the one first formulated by Fukuda and Komamine (1980), but vary at concentrations especially with the phytohormones. In this work equimolar concentrations (1mg/L each) of α-naphthalene acetic acid (NAA) and benzylaminopurine (BA) in the ICM gave the best results among other combinations.


Table 1.

Composition of culture medium used in establishing xylogenic Zinnia elegans mesophyll cell cultures.

Medium componentsConcentration (mg/L)
Macroelements
KNO32020
NH4Cl54
MgSO4·7H2O247
CaCl2·2H2O147
KH2PO468
Microelements
MnSO4·4H2O25
H3BO310
ZnSO4·7H2O10
Na2MoO4·2H2O0.25
CuSO4·5H2O0.025
Na2EDTA370
FeSO4·7H2O28
Organic growth factors
Glycine20
myo-inositol100
Nicotinic acid50
Pyridoxine-HCl0.5
Thiamin-HCl0.5
Biotin0.05
Folic Acid0.05
Phytohormones
Auxin (NAA)a1
Cytokinina1
Sucrose10 000
D-mannitol36 400
pH5.5
a Filter-sterilized and added after autoclaving the culture medium.

4.4 Cytological and biochemical measurements

Similar to developmental processes of many other cells, the in vitro TE differentiation can be subjected to cytological, biochemical and microscopical techniques. In this work, the following parameters were measured on the established cultures: cell viability, cellulose deposition, lignification of the cellulose fibres, density and integrity of nuclei, vacuolar collapse, DNA laddering (using TUNEL method), %TE and TE anatomy.

4.4.1 Vital staining with fluorescent diacetate (FDA)

The viability of the mesophyll cells in the culture is an important parameter both at the beginning and during differentiation of TEs. FDA vital staining is based on the detection of the esterase enzyme activity which is exclusively associated with living cells. These esterases lose their activity once the cell dies. Hydrolysis of FDA by the esterases in living cells produces an acetate moiety and a yellow florescein. Two drops each of cell suspension and aqueous FDA solution (0.01%w/v) were mixed on glass slide and observed with 20x or 40x objective of fluorescent microscope (Nikon Diphot) using excitation wavelength of 488nm. Only living cells appear yellowish green.

4.4.2 Nuclear condensation, DNA labelling by TUNEL and gel electrophoresis

The morphology of the nuclei in control cells and in the differentiating TEs was studied at 12h time intervals. Cells were first fixed in 4% formaldehyde (FA) solution containing 0.025% glutaraldehyde (GA), followed by staining with 1ng/ml 4,6-diamidino-2-phenylindole (DAPI) in 0.1% Triton X-100 (pH 4–6). The nuclei were examined with a Nikon Diaphot fluorescence microscope.

For in situ detection of nDNA fragmentation in cells undergoing xylogenesis, samples were collected at various time points and fixed in MSB buffer (100mM PIPES, 2.5mM EDTA, 2.5mM MgSO4·7H2O, pH 7.4) containing 4% FA and 0.025% GA for 1h at room temperature. The cells were immobilized on glass slides coated with poly-l-lysine and dried at room temperature. The fixed, dried cells were incubated for 20min at 37°C in a permeabilization solution containing proteinase K (20μl/mg proteinase K in 50nM Tris–HCl, pH 7.5, 1mg BSA). This was followed by air drying the area around immobilized cells that had been rinsed twice in PBS (200nM NaCl, 50nM Na2HPO4, 50nM NaH2PO4). TUNEL labelling was done by applying 50μl reaction solution to cells and incubating the slide in a humidified atmosphere at 37°C for 1h in the dark according to the manufacturer's instruction manual for suspension cells (Roche Applied Science, Germany). This TUNEL kit requires no further amplification of the signal (label). The samples were immersed in fresh MSB buffer (pH 6.9) and observed immediately with a Nikon fluorescence microscope at 510nm.

Isolation of nDNA from Zinnia suspension culture at various time points for DNA laddering detection with gel electrophoresis was performed according to De Jong et al. (2000) with slight modifications. About 1ml of pelleted and frozen mesophyll cells/TEs were ground into powder in liquid nitrogen. The powder was mixed with 15ml 65°C extraction buffer (0.1M Tris, 50Mm EDTA, 50mM NaCl) which was freshly mixed with 15μl β-mercaptoethanol and 1ml 20% SDS and incubated at 65°C for 20min. Then 5ml 5M K-acetate was added to the mixture, kept in ice for 30min and centrifuged at 4°C, 3500rpm for 30min. The supernatant was filtered through a tissue paper and collected in a new tube, mixed with one equal volume of isopropanol, and immediately spun down at 4°C for 5min. The pellet was briefly dried, dissolved in 300μl buffer (0.2M Tris pH=7.5, 50Mm EDTA, 2M NaCl, 2% cety-N,N,N triethyl ammonium bromide) and incubated at 65°C for 15min. The sample was extracted with one equal volume of chloroform. The supernatant was mixed with one equal volume of isopropanol in a new tube and centrifuged at 4°C for 5min. The pellet was dried and dissolved in 15μl TE buffer (10mM Tris pH=8.0, 1Mm EDTA). After measurement of DNA concentration, 0.1μgμl−1 RNase and 1.5μl loading buffer were added. Agarose gel (1.8% agarose) electrophoresis was performed with 15μg DNA per lane.

4.4.3 Staining of the cell wall

Cellulose deposition on the cells was measured by staining the cells or TEs with an aqueous Calcofluor white (CW) solution (0.0001%w/v) and slides observed under fluorescence microscope. CW shows fluorescence upon exposure to 365nm when bound to cellulose emits at 410nm (visible as white-blue). Care must be taken in order not to contaminate the cells with fungal hyphae or remnants from tissue paper on the slide as they contribute to the background noise.

4.4.4 Lignin measurement

Lignified TEs were detected by staining the lignin component with phloroglucinol-HCl (Siegel, 1953). Phloroglucinol is dissolved in 20% HCl, about 1% (w/v). Then a 1:1 mixture of the cell suspension and this solution are incubated on a glass slide for 10min at room temperature. This staining procedure stains lignin of the cell wall of TEs reddish purple. Alternatively, lignified TEs were observed as blue-white autofluorescence emanating from the lignified secondary thickening when observed under fluorescence microscope at 400nm wavelength.

4.4.5 Vacuolar staining

The intact vacuoles can be distinguished by staining with dyes that can enter the cytoplasm but are excluded from the vacuole. Here we stained the cells or TEs with FDA (0.01%w/v) and observed with fluorescence microscope using 488nm excitation wavelength. Cells with intact tonoplast (intact vacuole) excluded the yellowish dye from the vacuole thereby staining only the plasmamembrane and cytosol.

4.4.6 TE measurement

To be able to compare percentage TE differentiation of two or more cultures with different initial cell viability values, a correction factor must be included in the equation to calculate the actual %TE (%TEactual). Thus,where %TEactual is the actual yield of TE in the culture; α is the number of TEs counted in a representative fraction of the culture; β is the total number of differentiated and non-differentiated cells in the representative sample analyzed; and γ is the initial viability value of the culture.

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Received 3 January 2009; accepted 31 January 2009

doi:10.1016/j.cellbi.2009.01.019


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)