African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 5, Num. 3, 1997, pp. 253-258

Nucleolar and nucleolus organiser regions activity in tea as visualised by silver staining

F.N. Wachira and R.C. Muoki^1

Tea Research Foundation of Kenya, P.O. Box 820, Kericho, Kenya
^1 Botany Department, University of Nairobi, P. O. Box 30197, Nairobi, Kenya

(Received 23 September, 1996; accepted 12 August, 1997)

Code Number: CS97031
Sizes of Files:
    Text: 18.5K
    Graphics: Tables and line drawings (gif) - 14.6 

Nucleoli and Nucleolus regions (NORs) in diploid, triploid and tetraploid tea (Camellia sinensis L.) were stained using a simplified technique of silver staining based on the colloidal two-stage method. Nucleoli were visualised in interphase nuclei as black spots against a yellow background. The mode of nucleolar number corresponded to multiples of the somatic cell number and was a good marker for ploidy. Mitotic metaphase chromosomes were stained yellow to golden brown with strongly contrasting NORs appearing as black segments.

Key words: Camellia sinensis, silver staining, nucleolus organiser regions

RESUME

Les zones nucleolees et nucleoles (NORs) dans le the diploide, triploide et tetraploide (Camellia sinensis L.) etaient teintes en se servant d'une technique simplifie d'argentage basee sur la methode collodale a deux stades. Les nucleoles etaient visualisees en interphase de nucleis comme des taches noires sur font jaune. Le mode du nombre nucleolaire correspondait au nombre de cellules somatiques multiples et constituait un bon marqueur de ploidie. Des chromosomes mitotiques en metaphase etaient teintes de couleur jaune or a brune or contrastant avec les NORs qui apparaissent comme de segments noirs.

Mots Cles: Zones nucleolees actives, the camellia sinensis, argentage

INTRODUCTION

Cytogenetic investigations in tea (Camellia sinensis L.) have for a long time been confined to ploidy determination (Bezbaruah, 1971; Jayasurya and Govinderajulu, 1975; Kondo, 1977; Kulasegaram, 1980; Wachira, 1991). Such investigations have demonstrated that tea forms a stable polyploid series with a basic number of 15 chromosomes (Kondo, 1977; Shiyan and Dapeng, 1989; Wachira, 1991). Polyploids are, however, not very common (Bezbaruah,1970). Chromosome complements in tea have been shown to be generally homotypic and symmetrical in relative length and kinetochore position (Shiyan and Dapeng, 1989; Wachira et al., 1997). There is therefore a high risk of reversal of order for tea chromosomes particularly those of intermediate length (Wachira et al., 1997). The complements consists of near metacentric to submetacentric chromosomes (Bezbaruah, 1975; Shiyan and Dapeng, 1989; Wachira et al., 1997).

Chromosomes can however, be finely resolved beyond the recognised features of size, position of centromere, presence of secondary constriction and degree of heteropycnosis by differential staining. Silver staining has been widely used particularly in animal cells for the specific detection of nucleolar organiser regions (Ag-NORs) on chromosomes (Goodpasture and Bloom, 1975; Howell et al., 1975; Bloom and Goodpasture, 1976; Hizume et al., 1980; Howell and Black, 1980; Sato et al., 1980; Lacadena et al., 1984). Silver staining has, however, been applied to plant species on limited scale (Schubert et al., 1979; Hizume et al., 1980). Nucleolus organiser regions (NORs) and their locations have often been investigated by N-banding (Matsui and Sasaki, 1973; Matsui, 1974; Howell et al., 1975). Some authors have, however, pointed out that the specificity of the N-banding staining does not always correlate with the presence of NORs (Faust and Vogel, 1974; Pimpinelli et al., 1976; Gerlack, 1977), but experiments on in situ hybridisation have positively demonstrated that the familiar achromatic secondary constrictions on chromo-somes are associated with NORs which represent chromosomal locations of genes coding for 18s+28s ribosomal RNA (rRNA) (Pardue and Hsu, 1975).

Since the silver staining reaction of NORs can be considered as an indication of gene activity, it can be used to analyse gene activity at the ribosomal DNA sites with the conventional light microscope. Several authors (Miller et al., 1976; Howell 1977; Schmiady et al., 1979) have demonstrated that only those NORs that were functionally active during interphase are stained by silver (Ag-NORs) at the next mitotic metaphase. In this context, NOR activity is referred to as "potential activity", i.e., the ability of the silver stained mitotic NOR to be transcribed in the preceding or in the following interphases.

One salient feature noted in tea cytogenetic studies is that mitotic metaphase chromosomes lack predominant satellites (Bezbaruah, 1975; Wachira et al., 1997) which may indicate that tea has low activity of the NORs, a condition which makes its chromosomes fail to produce secondary constrictions. The present study was undertaken to detect the presence of NORs in the tea plant using an Ag-NOR method and to evaluate nucleolar polymorphisms in diploid, triploid and tetraploid tea.

MATERIALS AND METHODS

Actively growing shoot tips of some commercial diploid, triploid and tetraploid clones of tea were collected for use as a source of meristematic tissue. The 14 diploid (2n=2x=30) clones used in the study were (TN 14/3, CG28U864, BB35, SC20/13, SFS150, 31/8, S15/10, 6/8, 57/15, AHP 12/28, 303/231, 31/37, 54/40 and 11/26). Sixteen triploid clones (395/2, 76/1, 76/2, 76/3, 400/1, 392/2, 392/1, 371/1, 383/1, 52/1, 378/1, 85/1, 84/1, 84/2, 382/1 and 386/1) and two tetraploid clones (311/287 and 31/30) were also evaluated.

Nucleolus organiser regions were stained using a simplified technique of silver staining based upon the colloidal two stage method of Howell and Black (1980) as modified and described by Amstrong and Ford-Lloyd (1989). Shoot tips from the test clones were pre-treated in 0.002% hydroxyquinoline solution at 20 C for 3 hours then fixed in 1:3 glacial acetic acid: ethanol and washed in three changes of distilled water. The shoot tips were then hydrolysed in 1N HCl at 60 C for 8 minutes before being washed in three changes of distilled water.

Meristematic tissue was isolated from the shoot tips and a drop of 45% acetic acid pipetted onto it. The tissue was then macerated homogeneously. Two more drops of 45% acetic acid were added to the macerated specimens and a coverglass placed on. The specimen was squashed between the coverglass and glass slide with minimum lateral movement. The slide was flame dried and the coverglass rolled off by use of running deionised water. Four drops of 50% silver nitrate (AgNO3) in distilled water and two drops of colloidal developer (2g gelatine: 99ml distilled water: 1ml formic acid) were added onto the microscope glass slide containing the freshly prepared specimen.

The solutions (AgNO3 and developer) were mixed and covered with a coverglass. The specimen was left on an open bench until the solutions turned brown in colour. Excess stain solution was drained off by squashing between Whatman No.1 filter paper. The slide with specimen was than placed on a slide warmer which had been stabilised at 70 C. Within 30 seconds, the silver nitrate mixture turned yellow and within two minutes it became golden brown.

The total number of randomly selected cells scored for NORs and nucleoli per clone were 30. In all, 960 cells were evaluated.

RESULTS AND DISCUSSION

In interphase nuclei, active nucleoli were visualised as black spots against a yellow background. Detection of active nucleoli indicates that tea is not inherent in low activity of NORs. Nucleolar numbers in such interphase cells of diploid, triploid and tetraploid tea are shown in Figure 1. Diploid clones had 1-4 nucleoli per cell whilst the range was bigger for triploids (1-7). Tetraploid clones had 2-5 nucleoli per cell. In a large number of cases, nucleolar numbers were observed to correspond to multiples of the somatic cell number. The mode of the nucleolar number in diploids, triploids and tetraploids was therefore 2, 3 and 4, respectively (Table 1). Thus, the mode of nucleolar number per cell therefore reflects ploidy and may be used as a reliable marker for ploidy level particularly in situations where mitosis is infrequent. In garlic (Allium sativum L.), Amstrong and Ford-Lloyd (1989) proposed the use of nucleolar number as a marker for ploidy and also used nucleolar activity as a reliable marker for morphogenetic potential of callus. Increased number of nucleoli in some tea cells may be due to coincident NORs in squashed cells or promiscuous activity of NORs. According to Schubert and Wobus (1985), promiscuous activity of NORs cannot be ruled out. The increased number of nucleoli in some tea cells could not be a result of polysomy for NOR chromosomes as revealed by metaphase chromosomes.

TABLE 1

FIGURE 1 a-d. Ag stained cells of tea. (a) Nucleoli in interphase cells of diploid (2n=30) tea-Clone 6/8, (b) Nucleoli in the triploid Clone 76/1, (c) Nucleoii in the tetraploid Clone 31/30, and (d) Metaphase chromosomes stained with Agtelomeric Ag-NOR chromosomes are revealed (arrows). Bar indicates 10uM.

Metaphase chromosomes of diploid and triploid cells undergoing mitosis are shown in Figure 1d. The chromosomes stained yellow to light golden brown with strongly contrasting NORs appearing as black segments. Detection of NORs on tea chromosomes without the accompanying satellites may indicate that tea has chromosomes involved in NORs but which do not exhibit secondary constrictions. The presence of such chromosomes is itself not uncommon (Tsuchiya, 1964; Nakamura and Tsuchiya, 1982). The existence of NOR chromosomes in tea possessing microsatellites which cannot be seen on slide preparations cannot also be entirely discounted in the present and previous cytogenetic studies. Schlarbaum and Tsuchiya (1984) have reported that some plant chromosomes possess microsatellites which fail to be seen on conventional slide preparations due to the pretreatment procedures which tend to shorten the chromosomes. To verify this on tea chromosomes, electron microscopy studies are recommended.

Although much is already known about the structure and organisation of rDNA (Williams and Robbins, 1992) not much is known about the regulation of expression of the different gene clusters in species with several NORs. The few studies undertaken have shown that genetic regulation of expression of the different individual NORs in the same genome is very complex (Flavell and O'Dell, 1979; Lopez-Leon et al., 1995). Several studies on nucleolar competition (amphiplasty) in interspecific hybrids have also been undertaken (Lacadena et al., 1984; Vieira et al., 1990).

Similar studies in tea, however, require that chromosomes associated with NORs be unequivocally identified. The high risk of reversal of order of chromosomes of intermediate size in tea (Wachira et al., 1997) would, however make this difficult. Differential staining may be adopted for banding and identification of individual chromosomes.

ACKNOWLEDGEMENT

This paper is published with the permission of the Director, Tea Research Foundation of Kenya.

REFERENCES

Amstrong, S.A. and Ford-Lloyd, B.V. 1989. Contrasting nucleolar activity in callus of beet and garlic as visualized by a new silver staining technique. Cytologia 54:553-558.

Bezbaruah, H .P. 1970. Cytological investigations. Tea Research Association of North East India, Annual Report, pp 40.

Bezbaruah, H.P. 1971. Cytogenetical investigations of the family Theaceae; chromosome numbers in some Camellia species and allied genera. Caryologia 24: 421-426.

Bezbaruah, H.P. 1975. Cytological investigations of the family Theaceae. Cytology of triploid tea. Experimental Agriculture 11:17-22.

Bloom, S.E. and Goodpasture, C. 1976. An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. Human Genetics 34: 199-206.

Faust, J. and Vogel, W 1974. Are N-bands selective staining of specific heterochromatin? Nature 249:352-353.

Flavell, R.B. and O'Dell, M. 1979. The genetic control of nucleolus formation in wheat. Chromosoma (Berl.) 71:135-152.

Funaki, K., Matsui, S. and Sasaki, M. 1975. Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosoma (Berl.) 49:357-370.

Gerlack, W.L. 1977. N-banded karyotypes of wheat species. Chromosoma (Berl.) 62:49-56.

Goodpasture, C. and Bloom, S.E. 1975. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma (Berl.) 53:3740.

Hizume, M., Sato, S. and Tanaka, A. 1980. A highly reproducible method of nucleolus organizing regions staining in plants. Stain Technology 55:87-90.

Howell, W.M., Denton, T.E. and Diamond, J.R. 1975. Differential staining of the satellite regions of human acrocentric chromosomes- Experientia (Basel.) 31:260-262.

Howell, W.M. 1977. Visualization of ribosomal gene activity:Silver stains proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma (Berl.) 62:361-367.

Howell, W.M. and Black, D.A. 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia (Basel.) 36:1014-1015.

Jayasurya, P. and Govinderajulu, V. 1975. Chromosome numbers in some tea clones. The Planters Chronicle, July 1975 issue, pp. 185-186.

Kondo, K. 1977. Chromosome numbers in the genus Camellia. Biotropica 9:86-94.

Kulasegaram, S. 1980. Technical developments in tea production. Tea Quarterly 49:157-183.

Lacadena, J.R., Cermeno, M.C., Ovellana. J. and Santos, J.L. 1984. Evidence for wheat-rye nucleolar competition (amphiplasty) in triticale by silver-staining procedure. Theoretical and Applied Genetics 67:207- 213.

Lopez-Leon, M.D., Cabrero, J. and Camacho, J.P.M. 1995. Changes in NOR activity pattern in the presence of supernumerary heterochromatin in the grasshopper Eyprepocnemis plorans. Genome 38:68-74.

Matsui, S. and Sasaki, M. 1974. Differential staining of nucleolus organizers in mammalian chromosomes. Nature 246:148-150.

Matsui, S. 1974. Nucleolus organizer of Vicia faba chromosomes revealed by the N-banding technique. Japanese Journal of Genetics 49: 93-96.

Miller. O.J., Miller, D.A., Dev, V.G., Tantravahi, R. and Croce, C .M. 1976. Expression of human and suppression of mouse nucleolar organizer activity in mouse-human somatic cell hybrids. Proceedings of the National Academy of Sciences (U.S.A.) 73:4531-4535.

Nakamura, C. and Tsuchiya, T. 1982. Cytogenetics of alien addition trisomics in sugar beet. I. Meiotic chromosome behaviour in nematode- resistant trisomics. Biol. Zbl. (transl.) 101: 227-240.

Pardue, M.L. and Hsu, T.C. 1975. Location of 18s and 28s ribosomal genes on the chromosomes of the Indian muntjac. Journal of Cell Biology 64:251-254.

Pimpinelli, S., Santini, G. and Gatti, M. 1976. Characterization of Drosophila heterochromatin II. C- and N- banding. Chromosoma (Berl.) 57:377-386.

Sato, S., Huzume, M. and Kawamura, S. 1980. Relationship between secondary constrictions and nucleolus organizing regions in Allium sativum chromosomes. Protoplasma 105:7785.

Schlarbaum, S.E. and Tsuchiya, T 1984. A chromosome study of coast redwood, Sequoia sempervirens (D. Don) Endle. Silvae Genetica 32:56-62.

Schubert, I., Anastassova-Kristeva, M. and Rieger, R. 1979. Specificity of NOR staining in Vicia faba. Experimental Cell Research 120:433- 435.

Schubert, I. and Wobus, U. 1985. In situ hybridization confirms jumping nucleolus organizing regions in Allium. Chromosoma (Bell.) 92: 143-148.

Schmiady, H., Munke, M. and Sperling, K. 1979. Ag-staining of nucleolus organizer-regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity. Experimental Cell Research 121: 425-428.

Shiyan, C. and Dapeng, Y. 1989. Cytology studies on the polyploid tea. I. Study of Tea 9:119-126.

Tsuchiya, T. 1964. Chromosome observations and their uses in genetics and breeding in barley. Barley Genetics I. Proceedings of the 1st International Barley Genetics Symposium. Broekuizen, S., Dantuma, G., Lamberts, H. and Lange, W. (Eds.), pp. 116-150.

Wachira. F.N. 1991. Newly identified Kenyan polyploid tea strains. Tea 12:1O-13.

Wachira, F.N., Njagi, G.D.E. and Magambo, M.J.S. 1997. A comparative intraspecific karyotype analysis in some Kenyan tea cultivars (Camellia sinensis L.O. Kuntze). Discovery and Innovation (In press)

Williams, S M. and Robbins, L.G. 1992. Molecular genetic analysis of Drosophila rDNA arrays. Trends in Genetics 8:335-340.

Vieira, R., Queiroz, A.. Morais, L., Barao, A., Mello-Sampayo, T. and Viegas, W. 1990. Genetic control of IR nucleolus organizer region expression in the presence of wheat genomes. Genome 33:713-718.

Copyright 1997 The African Crop Science Society 

The following images related to this document are available:

Line drawing images