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Apart from germ line polyploidy in gametic cells, plants often exhibit endopolyploidy – genome variation in which multiple ploidy levels are found in somatic cells (Nagl, 1978; De Rocher et al., 1990; Joubès & Chevalier, 2000). Endoreduplication, the process of endonuclear chromosome duplication without intervening segregation and cytokinesis, is considered the most common mode of cell polyploidization in plants, being present in > 90% of angiosperms (Joubès & Chevalier, 2000). Endopolyploidy has been reported in many plant tissue types, especially those with cells of large size and high specificity. For orchids in particular, it has been observed in leaves (Jones & Kuehnle, 1998), root tips (Jones & Kuehnle, 1998), perianth (Mishiba & Mii, 2000) and growing embryos (Alvarez, 1968; Nagl, 1972).
Systemic control of endopolyploidy has been shown in plant species such as ice plant Mesembryanthemum crystallinum (De Rocher et al., 1990), Arabidopsis thaliana (Galbraith et al., 1991), cucumber (Gilissen et al., 1993) and tomato (Smulders et al., 1994). Increasing evidence has revealed that the pattern of endopolyploidy is characteristic of tissue type and developmental stage, indicating that endoreduplication is spatially and temporally regulated. In many plant species, older tissues showed higher levels of ploidy than younger ones (De Rocher et al., 1990; Galbraith et al., 1991; Smulders et al., 1994). It is suggested that endoreduplication is involved in developmental events such as cell expansion and differentiation (Kondorosi et al., 2000; Larkins et al., 2001). Furthermore, there is growing evidence that cell size is linked to the degree of endoreduplication (Melaragno et al., 1993; Cebolla et al., 1999; Kondorosi et al., 2000).
Specific combination of hormone, nutrient and light, which are major regulators of cell cycle and growth, might possibly initiate the signals that transform the mitotic cycle to endoreduplication during cell differentiation (Kondorosi et al., 2000). However, reports on the effects of plant growth regulators on endopolyploidy are equivocal and vary with plant species and tissue type investigated. Auxin treatment induced endoreduplication in tobacco single cells (Valente et al., 1998), but not in cactus Mammilaria san-angelensis cultured regenerants (Palomino et al., 1999). Enhancing effect of gibberellins on endoreduplication in Pisum sativa was cultivar-dependent (Mohamed & Bopp, 1980; Callebaut et al., 1982). Further investigation on the effects of auxins and gibberellins on endopolyploidy might provide more understanding of the role of these plant growth regulators in such a phenomenon.
Orchids are widely propagated for commercial purposes via tissue culture, with a variety of plant parts as explants (Arditti, 1996). A better insight to the plant genetic profile could be useful in tissue culture, since cytological differences inherent in explants or induced in regenerants during culture, might be some possible factors contributing to somaclonal variation. The objectives of this present study were to determine the occurrence of multiploid cells in different tissues of orchid Vanda Miss Joaquim and investigate whether the pattern of endopolyploidy could be affected by development. Effects of NAA and GA3 on endopolyploidy in the plant subject during embryo development were also investigated.
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Leaves of Vanda Miss Joaquim, mainly made up of large mesophyll cells, exhibited high endopolyploidy possibly to support the cell volume. De Rocher et al. (1990) had suggested that cells expected to reach high ploidy levels were large-volume cells such as water-storing mesophyll cells. Distribution of multiploid cells in Vanda Miss Joaquim leaves was coupled with development and often varied with leaf parts. Endopolyploidy seemed to be originally absent in the leaves and might have been initiated during the early stages of leaf development (Table 1). The endoreduplication process was apparently most rapid in growing young leaves, since nuclei of 9-cm long first leaf could possess DNA content up to 16C, as compared to the absence of endopolyploidy in 5-cm long first leaf (Table 1). Analyses on ninth and 16th leaves suggested that older leaf tissues exhibited higher ploidy levels than younger ones (Table 1), which were similar to the results from ice plant (De Rocher et al., 1990), tomato (Smulders et al., 1994) and Arabidopsis thaliana (Galbraith et al., 1991). Cell expansion could occur with age, thus a higher DNA content fulfilled by endoreduplication might be needed to sustain the larger cell volume. It supports the suggestion that endoreduplication is involved in cell development and differentiation (Kondorosi et al., 2000; Larkins et al., 2001). Besides, new rounds of endoreduplication might have started from leaf tip and progressed towards the base, since nuclei of higher C-levels in leaves always appeared first in the tip and last in the base during development (Table 1). It will be interesting to find out in future, the relative times leaf cells at the tip, middle and base switch from division to expansion. In algae Alaria esculenta sporangia, there is a series of four mitotic divisions in which there is only one round of DNA synthesis (Garbary & Clarke, 2002).
Observed absence of endopolyploidy in the shoot apex of Vanda Miss Joaquim (Table 1) further supported the proposal that endoreduplication had started during early leaf development, after the shoot stage. Endopolyploidy was absent in shoot tip of Brassica rapa and B. oleracea too and it was suggested that repression of endoreduplication at the apical meristem might be one mechanism to ensure the genetic stability of the germ line (Kudo & Kimura, 2001a). Also, cells at the apical meristem being small might not need a high nuclear DNA level to support their function.
In Vanda Miss Joaquim, only the tip of the aerial root exhibited endopolyploidy and its degree did not change significantly with root growth (Table 1). The pattern at the tip might not be due to the presence of large cells, as other root parts also have a substantial number of large cells. A more intricate differentiation control might be involved, since the aerial root tip is morphologically different from the other parts; for example, it is photosynthetic and not covered by veleman (Hew & Yong, 1997). By contrast, extensive endopolyploidy occurred in the whole of terrestrial roots (Table 1). In tomato roots, the proportion of 8C nuclei present increased upon arbuscular mycorrhizal colonization (Berta et al., 2000). Mycorrhizal infection is often associated with nuclear hypertrophy (Goh et al., 1992; Berta et al., 2000), which depends on chromatin decondensation and possibly endoreduplication (Berta et al., 2000). Terrestrial roots used for the present study were only mildly infected, thus fungal infection could not account for much of the extensive endopolyploidy. Besides, there is no direct evidence linking mycorrhizal infection and endoreduplication at present. Endoreduplication has been suggested to be involved in cell differentiation (Kondorosi et al., 2000; Larkins et al., 2001). Aerial and terrestrial roots of Vanda Miss Joaquim have distinctly different cell composition, morphology and growth characteristics, and they experience diverse environmental conditions. Whether these factors contribute to their different patterns of endopolyploidy remains to be elucidated.
Our findings indicated that endoreduplication was not a common feature in the cell growth and development of Vanda Miss Joaquim flowers and did not seem to couple with tissue differentiation in the flowers, with the column as an exception (Table 1). Endopolyploidy was also not detected in floral organs of Arabidopsis thaliana and it was suggested that the exclusion of multiploidy from somatic cells of floral structures might avoid the potential production of polyploid gametophytes (Galbraith et al., 1991). However, endoreduplication in perianth was reported in cabbage (Kudo & Kimura, 2001b) and portulaca (Mishiba & Mii, 2000). The column displayed an atypical pattern of endopolyploidy after anthesis with a substantial nuclei population with DNA content intermediate of 8C and 16C (Table 1), possibly contributed by its structural modifications. Confocal microscopic analysis of the column showed that it contained many cell types of diverse sizes and the nuclear DNA content was not highly correlated to cell size (W. L. Lim , unpublished data).
Endoreduplication occurred during embryo development (Table 2). Cytological studies on embryos of another orchid, Vanda sanderiana, revealed that while nuclei in the meristematic cells remained 2C throughout development, endopolyploidy was exhibited in the parenchymal cells (Alvarez, 1968). Endoreduplication in parenchymal cells of growing embryos had also been implicated in Cymbedium (Nagl, 1972) and Spathoglottis plicata (Raghaven & Goh, 1994). Confocal microscopic analysis of Vanda Miss Joaquim protocorm showed that parenchymal cells had very much larger cell and nuclear areas than meristematic cells, indicating that endopolyploidy might be a very prominent feature in the former (W. L. Lim, unpublished data). Orchid seeds lack endosperm and cells present were heavily packed with food reserves (Arditti, 1992). The parenchymatous region of orchid embryo appears to be analogous to the endosperm of other angiosperms, in that both tissues function in the nutrition of the embryo (Alvarez, 1968). During maize endosperm development, endoreduplication occurred to drive massive synthesis of storage proteins and starch (Lur & Setter, 1993). The degree of endopolyploidy increased with embryo development of Vanda Miss Joaquim (Table 2), possibly therefore to support the nutrient requirements of growing protocorms.
Exogenous NAA might increase the endopolyploidy levels in embryos of Vanda Miss Joaquim (Table 3), though it was observed that the rate of embryo growth decreased with increasing NAA level. Auxin was reported to affect endosperm development in maize, which involved endoreduplication (Lur & Setter, 1993). At present, auxin has induced endoreduplication in culture for most of the studies at varying extents (Valente et al., 1998; Gendreau et al., 1999; Mishiba et al., 2001), though in Mammillaria cactus, no such enhancing effect of auxin was observed (Palomino et al., 1999). This suggested the possible involvement of auxins in the regulation of endoreduplication. Evidence indicated that inhibition of the activity of p34cdc2, a cell cycle regulator, might result in the switch of mitotic cycles to endoreduplication and this inhibition might involve auxin (John et al., 1993; Valente et al., 1998).
Only 10−5 M GA3 caused a small increase in the amount of endoreduplication for the embryos after 4 weeks of culture (Table 4). It was also observed that only 10−5 M GA3 led to a mild induction in embryo growth. This indicated that GA3 might induce endoreduplication and growth in Vanda Miss Joaquim embryos by a small extent, with its effect being dependent on concentration. GA treatment increased the endopolyploidy levels in GA-deficient mutants of Arabidopsis (Gendreau et al., 1999). In addition, the enhancing effect of GA on endopolyploidy appeared to be cultivar-dependent in Pisum sativa (Mohamed & Bopp, 1980; Callebaut et al., 1982) and Triticum durum (Cavallini et al., 1995).
There is growing evidence that, in context of a given genome, the size and morphology of cells are linked to their DNA content (Kondorosi et al., 2000). Cells that have undergone endoreduplication are larger than comparable cells that have not (Larkins et al., 2001). Studies on Triticum durum and Arabidopsis thaliana epidermal cells also showed the correlation of cell size and nuclear DNA content (Cionini et al., 1983; Melaragno et al., 1993). Recent work had provided molecular evidence for cell size regulation by endopolyploidy. One gene involved in the transformation of mitotic cycles to endoreduplication, ccs52, was identified in alfalfa. Reduction in ccs52 transcript level correlated with a decrease in endopolyploidy and cell size in Medicago roots, cotyledons and hypocotyls (Cebolla et al., 1999). Positive correlation between cell volume and degree of endopolyploidy in most cell types indicates that nuclear DNA content might play a key role in regulating cell volume. This could also apply to Vanda Miss Joaquim.
In conclusion, systemic control of endopolyploidy was displayed where multiploid cells were found in many tissues of Vanda Miss Joaquim with varying patterns. Our results showed that embryos of Vanda Miss Joaquim were originally diploid and that extensive endoreduplication occurred during embryo development (Table 2; Fig. 2). Such a high degree of endopolyploidy was replaced by the differential distribution of multiploid cells in organs of the mature plant (Table 1). The pattern of endopolyploidy was found to change with development for embryos, leaves and flowers (Tables 1 and 2). These findings are in line with the general observation that the distribution of endopolyploid cells is characteristic with tissue type and developmental stage. Organ-specificity in the pattern of endopolyploidy in Vanda Miss Joaquim might possibly reflect its integral involvement in cell expansion and differentiation. Endoreduplication was suggested to cope with the gene expression required for cell expansion and specialization, rapid cell growth, high metabolic activity, coordination with organelle genomes, or adaptation to environmental changes (Galbraith et al., 1991; Joubès & Chevalier, 2000; Larkins et al., 2001).
Somaclonal variation can arise from genetic changes such as gene mutation (Dennis et al., 1987), increase in chromosome number (Lewis-Smith et al., 1990) and DNA methylation (Jaligot et al., 2000). Apart from elucidating the gene expression during differentiation and development, studies on endopolyploidy could also give understanding to the nature of tissues used for clonal propagation, thus providing some possible explanations to the occurrence of somaclonal variation. Plant regeneration is based on the concept of cell totipotency. An explant exhibiting endopolyploidy would contain a mixture of cells of varying ploidy levels, therefore cell heterogeneity in DNA content within the explant might contribute to variation in gene expression and phenotype of somaclones. Vanda Miss Joaquim could be micropropagated using shoot tip, stem, axillary buds, leaf and root tip explants (Arditti, 1996) and the last two have been shown to exhibit a high degree of endopolyploidy (Table 1). If genetically uniform plants were to be micropropagated, explants could be obtained from organs in which endopolyploidy is absent, such as shoot tip for Vanda Miss Joaquim, so to minimize variation. Apart from the endopolyploid nature of original explant, the presence of plant growth regulators in culture might induce or inhibit endoreduplication and alter gene expression of cultured plants, as seen in Vanda Miss Joaquim (Tables 3 and 4). Hence, knowledge on the effect of plant growth regulators on endoreduplication would be helpful in the maintenance of ploidy levels of cultured plants.