Shoot meristem of many flowering plant species consists of three independent cell layers (L1, L2 and L3) (Tilney-Bassett, 1986). The cells in the two outer layers (L1, L2) divide anticlinally in the same layer while those in inner layer (L3) divide in any plane (Barton et al., 1993; Li, 2005). If the genotypes of cells at least two layers in a plant meristem are different, the plant is called chimera (Tilney-Bassett, 1986; Burge et al., 2002). When one or more entire cell layer(s) are genetically different from other cell layers, such a chimera is called periclinal chimera; when a sector of all cell layers of a chimera is genetically different, it is called sectorial chimera; mericlinal chimera exists when a fraction of one or more cell layers (but not the entire layer) of a shoot apical meristem is genetically distinct from the remainder of the layer. Periclinal chimera is the most stable form, and it can produce distinctive and valuable plant phenotypes for horticulturalists. The three apical layer of periclinal chimera with two genotypes through translocation can produce 2³ different arrangements with six periclinal chimeras (Tian et al., 1993; Li et al., 2005).

A chimera with different ploidy levels in the apical layers is cytochimera (Tilney-Bassett, 1986). The cytochimera is important to analyse the growth relationship between the cell layers of different ploidy levels and to study the biochemical interaction between cells with different genotypes (Sessions et al., 2000; Marcotrigiano, 2001).

The studies of the chimeras showed that the cells of outermost shoot apical layer (L1) normally give rise to the plant epidermis (Carpenter et al., 1995; Li et al., 2005). The cells in the second apical layer (L2) differentiate themselves as subepidermal tissue, e.g. palisade parenchyma, the spongy parenchyma of the leaf margin, outer part of the cortex, as well as gametes (Burge et al., 2002). Cells in the L3 derive mainly into the central tissues (e.g., pith) and roots of cutting(Tilney-Bassett, 1986). Therefore, conventional squeezing-method with shoot for determining of chromosome number can not be used in cytochimeral tissues, as the cells of all layers in the shoot tips will be mixed together by squeezing; subsequently the cell layers are not easy to be distinguished from each other.

Adaniya et al. (1994) presented a method for indirect detection of cytochimeras with the maximum number of nucleoli per cell by stomata and pollen. Zonneveld et al. (2000) and Burge et al. (2002) reported that the cytochimeral tissue could be determined with the modern flow cytometry by analysis of nuclear DNA content of different cells in the cytochimera, but it is difficult to distinguish the exact layers. Mochida et al. (2004) had successfully captured the chromosomes and mitosis in hybrid embryos of wheat and maize under confocal scanning optical microscopy, but it is too expensive for most experiments.

Zonneveld et al. (2000) reported that higher ploidy levels usually result in proportionally larger cells. Jovtchev et al. (2006) confirmed that nuclear DNA content, nuclear volume and cell volume are positively correlated in angiosperms. Therefore, observations of cell size in longitudinal sections through the apex are used usually to determine the ploidy level of the different layers in cytochimeras. Usually the comparisons of epidermal cell size and pollen size of the cytochimera are used to indirectly determine the cell ploidy levels in L1 and L2 layers. The chromosome number of root tips is used to indicate the ploidy levels of L3, but the conclusion should be carefully drawn because the cells in root tips are easily to mutate(Zonneveld et al., 2000). To my knowledge, there is not any published report for direct determining ploidy of the somatic cells in different layers of a cytochimera. This work developed thus a method for easy, direct and precise determining of the ploidy levels of the somatic cells in different layers of cytochimeral tissues.

1 Materials and Methods

The cytochimeral mutant clone HDD (L1 was haploid; L2 and L3 were diploid) and the clone 5/74/2 from the haploid Pelargonium zonale 'little lady' were used to examine the ploidy levels of somatic cells in each layer. 1.1 Tissue collection and pre-treatment The terminal of a mitotically active shoot was collected from established plants between 9:00 am -14:00 pm in sunshine. The younger outside leaves of the shoot tip were carefully removed in the laboratory under a microscope (Cytoplaten). The terminal shoot apices were soaked in 0.1% colchicine solutions for 6 h in dark at 4 ℃ in order to availablely spread the chromosomes for study. Then the gas in the shoot tips were exhausted with a pump for 15 min in ethanol-chloroform-acetic acid (6:3:1, V/V/V) mixture and then the shoot tips were kept in the solution for 24 h at an ambient temperature.

1.2 Draining and Embedding

The water in the cells of the shoot tips was exchanged sequentially in ethyleneglycol-monoethyl-ether, 96% ethanol, propane and butane, each for 24 h. Then the shoot tips were polymerized for 24 h in the dark in a solution containing 100 mL Technovit 7100 (GMA with co-catalyst X-Cl; Germany) and 1 g dibenzoylperoxid. Then the shoot tips were carefully placed in an embedding capsule with embedding mixture which (10 mL above solution and 1 mL dimethylsulfoxid) allow solidifying.

1.3 Section preparation and staining

The embedded shoot tips were sectioned to 8~10 μm with a rotation microtome. The sections were carefully floated in series on a glass slide with a drop of water. After drying the sections on the slides, the sections were stained for 10 seconds in Toluidin-Blue-Solution (Böck, 1989): 60 mL 1% Toluidin blue water solution was mixed with 30 mL 2.5% sodium-carbonate water solution and 10 mL 70% alcohol. Then the sections of shoot tip were washed with running water. After drying the stained sections on a hot plate at 60 ℃, the good stained sections on the slides were covered with a cover slip using DPX solution (Fluka).

2 Results 2.1 Effect of staining

The processes of mitotic nuclear division in the somatic cells in investigated tissue of the P. zonale 'little lady' were observed by this method (Plate Ⅰ-1~4). The Toluidin-blue could not only stain intensively the chromosomes with a very clear figure, but they also could dye the spindle sufficiently with a good contrast for photography. It showed that this method is right for determining the ploidy levels with the chromosome numbers and for investigating the nuclear division in muted cells.

2.2 Ploidy determination for cytochimeral tissues

The cell size in outermost layer (L1) of the longitudinal section of meristem of the cytochimeral plant HDD was smaller than the cell sizes in L2 and in L3 (Plate Ⅰ-5). The epidermal cells in the leaf primodium section contained 9 chromosomes (Plate Ⅰ-6), which is the basic number of chromosomes for a typical haploid plant 'little lady' of P. zonale. The cells in subepidermal layer and inner layer of leaf primodium sections however, contained only diploid chromosome numbers (Plate Ⅰ-7, 8). The chromosome numbers in different layer cells proved that such a cytochimera was a haploid-diploid-diploid constitution. The investigation showed that the chromosome numbers of the cell layers of the leaf primodium sections corresponded with the ploidy levels of the cell layers of the meristem.

The cell size in the outermost layer of longitudinal section of the meristem of clone 5/74/2 was bigger than the cell sizes in L2 and in L3 (Plate Ⅰ-9). The cells in epidermis in leaf primodium section of clone 5/74/2 contained a tetraploid chromosome number (Plate Ⅰ-10). The cells in the subepidermal layer and inner layer of leaf primodium sections of clone 5/74/2 had only haploid chromosomes (Plate Ⅰ-11, 12). The investigation showed that clone 5/74/2 had a cytochimeral constitution with genotype tetraploid-haploid-haploid.

3 Discussion

This method combines the investigation of the histology with the cytological analysis. It is not only able to determine the ploidy of the different cell layers in the cytochimera (Plate Ⅰ-5~12), but had also observed the mitotic division of the somatic cells (Plate Ⅰ-1~4). It was the first time to exactly determine the ploidy of cytochimeral clone 5/74/2. In additional, the irregular nuclear divisions were observed in the muted epidermal cells of the clone 5/74/2 (not published). Therefore, this method cannot only be used to directly determine the ploidy levels by counting the chromosome number of the somatic cells in different layer in the cytochimeral tissue, but can also be used for the study of cell division procedures of the mutated cells in any tissue. Of course, the series of the section during counting must be taken into consideration to avoid incorrect chromosome number counting.

The investigations of the chromosome number of the cells in leaf primodium sections supported the developmental anatomy of apical stable periclinal chimeras by Clark (1997) that the three independent apical layers can be traced in stem, leaf and flower. Although the appearance of ploidy mutation and flexibility could occur during the developing of the leaf tissue, the chance of the spontaneous mutation was relatively small. Therefore, the number of chromosomes in leaf tissue could be used to indicate the ploidy level of correlated cell layers in shoot apices or axillary's buds. Furthermore, the cells of the leaf primodium section are better materials to be used for chromosome observation than the cells of the shoot apical section.