_??_1989 by Cytologia, Tokyo Cytologia 54: 553 -558 , 1989

Contrasting Nucleolar Activity in Callus of Beet and Garlic as Visualised by a New Silver Staining Technique

S. A. Armstrong1 and B. V. Ford-Lloyd2

1 Regional Cytogenetics Unit, East Birmingham Hospital , Bordesley Green, Birmingham, UK 2 Dept. of Plant Biology, University of Birmingham , PO Box 363, Birmingham BI5 2TT, UK Accepted August 8, 1988

Successful regeneration of whole plants from callus is dependent upon species and often genotype, as well as upon the presence of growth regulators and medium composition. This is exemplified particularly in beet (Beta vulgaris L.) where the tissue or organs from which re sponsive callus is produced, and particularly the genotype governs the success of regeneration (De Greef and Jacobs 1979, Saunders and Daub 1984, Ford-Lloyd and Bhat 1986). The medium composition, particularly with respect to growth regulators has complete influence on callus production and its ultimate organogenic capacity. This situation contrasts with that of garlic (Allium sativum L.) where the potential for plant regeneration from callus is far less dependent on genotype, or the means by which callus is produced, and where callus is capable of switching to a regeneration phase after extended periods of culture confined to callus proliferation (Khadzir 1987). We have assessed the cytological activity of callus from different sources for nucleolar activity with a view to obtaining a reliable marker for regeneration potential of callus. This we have attempted by studying nucleoli in interphase nuclei of callus cells, using a simplified technique of silver staining based upon the colloidal two-stage method of Howell and Black (1980). This technique can be used to assess the numbers of nucleoli occurring in cells and also for detecting nucleolar organiser regions (NORs) in cells in mitotic metaphase.

Materials and methods

Production of callus: Callus (Frll) was initiated on basal plate explants of cloves of garlic cv. Fructidor using MS medium (Murashige and Skoog 1962) with 3 mg 1-1 2, 4-D (Kehr and Schaeffer 1976). Callus was either subcultured every 28-42 d on the same medium for maintenance of callus (without organogenesis), or (Fr30) onto MS medium containing IAA and kinetin (1 mg 1-1) which induced continued regeneration of shoots accompanied by callus growth (Kehr and Schaeffer 1976). Four beet genotypes were selected because of their known response to in vitro culture (Bhat 1986). Two genotypes, SKE/11 and B382/2 were ones selected from sugar beet cv. Sharpe's Klein E and cv. Bush Mono respectively, which were capable of producing shoot culture callus with the capacity to regenerate (Bhat 1986). The other two genotypes, B1064/66 and B1064/103 were both of the fodder beet cv. White Knight, and formed callus on shoot cultures which was not responsive in terms of regeneration. Of the four genotypes, new shoot cultures were established from stock shoot cultures on MS medium with 0.25 g 1-1 BAP (Saunders and Daub 1984), and cytological observation was then made on the callus which formed around the base of the cultures after 42-56 d. Cytological examination was also carried out on callus of this origin 14 d alter subculture onto 3 different media chosen to effect different cultural responses: MS medium supplemented with BAP, BAP plus IBA, and IBA (each at 0.25mg 1-1). 554 Armstrong, S. A. and B. V. Ford-Lloyd Cytologia 54

Silver staining: Small pieces of callus were harvested, placed on glass slides, tapped out and squashed in a drop of 45% acetic acid. Cover slips were removed using a freezing spray. For staining, 4 drops of 50% silver nitrate were placed on a slide, followed by 2 drops of col loidal developer (2g gelatine: 99ml distilled water: 1ml formic acid). A cover slip was added and the slide placed on a hot plate at 65-70•Ž for 2-3 minutes until the preparation turned brown. Alternatively, the slide was placed above the water in a water bath at 60•Ž. The cover slips were floated off under tap water, and the material counterstained with 4%

Fig. 1.

Giemsa in Sorensen's phosphate buffer for 1 minute. When using the moist chamber the process was slightly slower, but had the advantage that staining could be monitored more easily.

Results

Metaphase chromosomes of a cell undergoing mitosis in garlic callus are shown in Figure 1989 Contrasting Nucleolar Activity in Callus of Beet and Garlic 555

Ia. The chromosomes stain light blue with Giemsa, and the NORs are strongly contrasting, appearing as black segments. Nucleoli can be visualised in interphase nuclei as black areas against a light blue background. Examination of the two types of callus of garlic maintained on different media indicated a marked difference in the ranges of numbers of nucleoli occurring in cells (Fig. 2). Cells from callus (Frll) grown solely on a callus maintenance medium (non-regenerating) yielded counts of between 3 and 15 or more, the mode being 6 (Fig. Ib). For the callus (Fr30) which had been subcultured to a medium inducing regeneration, the range was much smaller (1-9) with a mode of 4 (Fig. lc). This latter mode was found to correspond to the normal nucleolar num ber of garlic somatic cells. It should be noted that nucleolar numbers per cell which do not correspond to multiples of the somatic cell number (2n=16) do not necessarily indicate an eusomaty, although this cannot be ruled out in the case of garlic (Novak 1981), rather that nucleoli are coincident in some of the squashed cells.

Fig. 2.

The beet shoot culture callus grown on different media and from regenerable versus non-regenerable genotypes could not be clearly separated into two groups in the same way as the garlic. Nucleolar numbers per cell in callus of all genotypes, and before and after sub culturing onto media with different growth regulator regimes showed no significant variation when data were subjected to a skew analysis or a chi-square analysis of means (p>0.99). The overall mean was found to be 1.175 nucleoli per cell. All callus was composed pre dominantly of groups of large vacuolated cells showing no nucleolar activity, but interspersed with smaller cells possessing mainly one but occasionally more stained nucleoli (Fig. ld). This was found to be similar to the number in normal root tissue, where the nucleolar number was predominantly one per cell, even regardless of the ploidy of the material (i.e. diploid or triploid). The ratios of vacuolated and small cells did not vary significantly between calli growing on different media and of different genotypes and were in the region of 200:1. Im mediately after subculture onto fresh medium however, the numbers of small cells showing 556 Armstrong, S. A. and B. V. Ford-Lloyd Cytologia 54 nucleolar staining generally increased to give a ratio of large to small in the order of 100:1. There was also a slight but statistically insignificant increase in the number of cells having more than one nucleolus (Fig. 3).

Discussion

Nucleolus organiser regions (NORs) of chromosomes appear as unstained areas on con ventionally stained chromosomes and are also known as secondary constrictions. These regions have been shown to contain multiple copies of genes that code for ribosomal RNA (rRNA) (Henderson et al. 1982). Both NORs and nucleoli can be visualised under light microscopy by silver staining because silver preferentially reacts with acidic proteins that bind to newly synthesised rRNA. Therefore active nucleoli, and NORs which were functionally active during the preceding interphase, are detectable by silver staining (Goodpasture and Bloom 1975, Miller et al. 1976, Howell 1977).

Fig. 3.

Consequently silver staining is recognised as being highly specific for the NOR. It was first used by Howell et al. (1975) to selectively stain NORs situated on the human acrocentric chromosomes. Howell and Black (1980) introduced an improvement to the earlier technique for mammalian cells by using a two-step method including a colloidal developer. This tech nique considerably speeds up the staining process, from several hours to a few minutes. Silver staining has been developed for use with several plant species (Hizume et al. 1980), but the protocols are lengthy and the results are generally inferior to those which can be obtained using mammalian chromosome preparations. The technique we have developed and described represents an improved and simplified procedure suitable for application to plant cells in general, and specifically in this case to plant cells in culture. Its use as a tool for identifying morphogenetically competent callus is also demonstrated for garlic where morphogenetic potential of callus is normally maintained over 1989 Contrasting Nucleolar Activity in Callus of Beet and Garlic 557

extended periods of time. Here, callus maintained in either a regenerating or non-regenerating form is found to be composed of a large proportion of cells possessing active nucleoli. The presence of active nucleoli in varying numbers indicates not only active morphogenesis on suitable medium, but also the maintenance of potential for morphogenesis under culture conditions not conductive to immediate regeneration of plants. This contrasts with beet however, where no clear indication of morphogenetic potential was given by nucleolar activity in cells. It may be possible to use nucleolar visualisation as an indicator of ploidy level in callus cells, in situations where mitosis is infrequent. It is clear from our study that increased num bers of nucleoli could be a result of polysomy for NOR chromosomes. However, it cannot be ruled out that some form of promiscuous activity of NORs (Schubert and Wobus 1985) is stimulated by in vitro culture, which could in turn lead to somaclonal variation amongst re generants. In the case of regenerating and non-regenerating garlic callus, the range of ploidy is very different, and appears to diverge far more from the diploid state in non-differentiating callus compared to that undergoing regeneration. Only in the case of garlic, was the number of cells with active nucleoli, as visualised by silver staining, an indicator of the actual or potential morphogenesis in actively growing callus. In certain cases therefore, nucleolar number may also serve as a marker for the type of cell division which leads to the formation of the particular cells required for meristem formation (Thorpe 1982), as opposed to cell division responsible for the normal maintenance and growth of viable callus (Gautheret 1966).

Summary

Silver staining procedure has been modified to provide a rapid means of visualising nu cleoli and NORs in a range of plant tissue including callus. Unlike the situation with beet, garlic callus was found to be composed of large numbers of cells possessing stained nucleoli. The number of nucleoli per cell varied depending upon the growth medium and hormone composition used for culture. On medium promoting organogenesis and subsequent plant regeneration, the nucleolar number per cell was lower than that in callus on non-regeneration medium, and similar to that in normal root tissue. Nucleolar number is therefore a marker for regeneration in callus. No such variation was found in beet, where no marker for regenera tion was found.

References

Bhat, S. 1986. Tissue and protoplast culture of beet (Beta vulgaris L.) aimed at plant improvement. Ph. D thesis, University of Birmingham, UK. De Greef, W. and Jacobs, M. 1979. In vitro culture of sugar beet: description of a cell line with high regeneration capacity. P1. Sci. Lett. 17: 55-61. Ford-Lloyd, B. V. and Bhat, S. 1986. Problems and prospects for the use of protoplasts in beet breeding. In Horn, W., Jensen, C. J., Odenbach, W. and Schieder, O. (eds.). Genetic Manipulation in Plant Breeding. De Gruyter, Berlin, pp. 437-440. Gautheret, R. J. 1966. Factors affecting differentiation of plant tissues grown in vitro. In Beermann, W. (ed). Cell Differentiation and Morphogenesis. North Holland Publishing Co., Amsterdam, pp. 55-71. Goodpasture. C. and Bloom, S. E. 1975. Visualisation of nucleolus organiser regions in mammalian chromo somes using silver staining. Chromosoma (Berl.) 53: 37-50. Henderson, A. S., Warburton, D. and Atwood, K. C. 1982. Location of ribosomal DNA in the human chromo some complement. Proc. Nat. Acad. Sci. USA 69:.1394-3398. Hizunie, M., Sato. S. and Tanaka, A. 1980. A highly reproducible method of nucleolus organising regions staining in plants. Stain Technol. 55: 87-90. 558 Armstrong, S. A. and B. V. Ford-Lloyd Cytologia 54

Howell, W. M. 1977. Visualisation of ribosomal gene activity: silver stains proteins associated with rRNA transcribed from oocyte chromosomes. Chromosoma (Berl.) 62: 361-367. - , Denton, T. E. and Diamond, J. R. 1975. Differential staining of the satellite regions of human acrocentric chromosomes. Experientia 31: 260-262. - and Black, D. A. 1980, Controlled silver-staining of nucleolus organiser regions with a protective colloidal developer: a one step method. Experientia 36: 1014-1015. Kehr, A. E. and Schaefer, G. W. 1976. Tissue culture and differentiation of garlic. Hort. Sci. 11: 422-423. Khadzir, B. D. 1987. Assessment of somaclonal variation in garlic (Allium sativum L.) tissue cultures by using isozyme markers. MSc thesis, University of Birmingham, UK. Miller, D. A., Dev. V. G., Tantravahi, R. and Miller, O. J. 1976. Suppression of human nucleolus organiser activity in mouse-human hybrid cells. Exp. Cell Res. 101: 235-234. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cul tures. Physiol. Plant. 15: 473-497. Novak, F. J. 1981. Chromosomal characteristics of long-term callus cultures of Allium sativum L. Cytologia (Tokyo) 46: 371-379. Saunders, J. W. and Daub, M. E. 1984. Shoot regeneration from hormone autonomous callus from shoot cul tures of several sugar beet (Beta vulgaris L.) genotypes. Pl. Sci. Lett. 34: 219-223. Schubert, I. and Wobus, U. 1985. In situ hybridisation confirms jumping nucleolus organising regions in Allium. Chromosoma (Berl) 92: 143-148. - ; Ohle, H., and Hanelt, P. 1983. Phylogenetic conclusions from Giemsa banding and NOR staining in top onions. Pl. Syst. Evol. 143: 245-256. Thorpe, A. T. 1982. Callus organisation and de novo formation of shoots, roots and embryos in vitro. In Tomes, D. T., Ellis, B. E., Harney, P. M., Kasha, K. J. and Peterson, R. L. (eds), University of Guelph, Guelph, pp. 115-138.