INVESTIGATIONS ON PLOIDY LEVELS OF HAPLOID AND DIPLOID CALLUS AND CELL SUSPENSION CULTURES OF DATURA INNOXIA MILL.

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1 J. Cell Set (1980) 365 Printed in Great Britain Company of Biologitts Limited ig8o INVESTIGATIONS ON PLOIDY LEVELS OF HAPLOID AND DIPLOID CALLUS AND CELL SUSPENSION CULTURES OF DATURA INNOXIA MILL. R. KIBLER, J. BLASCHKE, E. FORCHE AND K.-H. NEUMANN InstitutfUr Pflanzenernahrung, Abt. Getoebekultur, der Justus-Liebig-Universitdt Giessen, Bundesrepublik Deutschland SUMMARY Using haploid and diploid Datura imtoxia Mill, callus cultures and cell suspensions it was shown that meristematic material usually attains the lowest possible C-value of a given ploidy level. Parenchyma material of callus cultures, however, indicates a broad scattering of C-values up to 16 C, which is paralleled by C-value distribution of the free cell fraction in actively dividing cell suspensions. In these suspensions, cell division activity seems to be restricted to small meristem-like clusters. INTRODUCTION In a number of publications (Vanzulli, Magnien & Olivi, 1980; for review see Sunderland, 1973) cytogenetic instability of tissue and cell cultures of higher plants was reported, mainly due to polyploidy, aneuploidy or chromosomal aberrations. The occurrence of these disturbances of the genetic system renders the use of such cultures rather difficult in genetic as well as molecular biological studies and limits their potential use in plant breeding. In a series of experiments a systematic study on C-value distribution in haploid and diploid callus and suspension cultures of Datura innoxia as influenced by kinetin were carried out to determine the course of ploidy pattern this material develops during a culture period of 4 weeks. The results of these investigations are summarized in this paper, their application to obtain ploidystable cell suspension cultures is published elsewhere (Kibler, 1978; Neumann, Blaschke, Forche & Kibler, 1978). MATERIALS AND METHODS Haploid Datura innoxia plants from which the primary explants were obtained were raised using a method of anther cultures as described by Forche & Neumann (1977), and diploid plants were raised from seeds. Care was taken at explantation that plants of both ploidy levels were in comparable developmental states. The aseptically obtained explants were cultured (22 C C, continuous illumination of ca lx on a phytostat) for 28 days in liquid cultures using a nutrient solution as described by Neumann (1965). In order to obtain highly viable cell suspensions of free-floating cell material, after 16 days of culture the contents of the culture vessels were passed through a polyester

2 366 R. Kibler, J. Blaschke, E. Forche and K.-H. Neumann filter (250 /im mesh), the filtrate was concentrated by centrifugation at 100 g, and after dilution with fresh medium (1:5; v/v) transferred to culture vessels eventually containing 15 ml of cell suspension (ca. io 4 cells/ml). The growth conditions were the same as during callus culture. Fresh weight and the number of cells were determined as published elsewhere (Kibler, 1978), and estimation of DNA content of callus material was carried out according to Blaschke, Forche & Neumann (1977) using a microfluorometric method. However, some modifications of the original method were required for DNA measurements of cell suspensions (For detail see Kibler, 1978, and Kibler, Forche & Neumann, 1979). RESULTS AND DISCUSSION As in other tissue culture systems during culture of freshly cut explants of Datura innoxia in a liquid nutrient medium after initiation of growth by cell division, cells and small cell clusters dissociate from the developing callus and float freely in the medium. In Fig. 1 the influence of kinetin on fresh weight of callus material and on the number of cells in cell suspension, partly organized in small clusters of up to about 500 cells, of haploid and diploid Datura calluses are summarized. As can be seen, no significant differences between the two ploidy levels can be observed with 10* = : io!_ I I Time in culture, days Fig. 1. The influence of kinetin on callus cultures of haploid and diploid Datura innoxia (fresh wt and development of' free' cells)., A, 2n without and with kinetin, respectively;» A, n without and with kinetin, respectively. I 24 I 28

3 Ploidy levels of haploid and diploid Datura cultures 367 respect to fesh weight production of callus material or to the number of floating cells. However, as in other tissue-culture systems (e.g. the carrot) a significant influence of kinetin on callus growth was found from about the tenth day after inoculation onward. In the plus-kinetin treatments, log phase lasts up to about the twentieth day whereas in minus-kinetin treatments the transition into the stationary phase can be already observed after 14 days of culture. The decrease in fresh weight of both treatments, i.e. plus- and minus-kinetin, during stationary phase of fresh weight production, is due to a high degree of sloughing off of cells from the periphery of the callus. The accumulation of floating cell material can be noted from the tenth ( kin) to the fourteenth ( + kin) day after the beginning of the experiment and coincides with the log phase of callus growth. In spite of the influence of kinetin on fresh weight, no significant changes in the number of cells per ml in cell suspensions was observed due to the kinetin supplement. After proper separation this free-floating cell material was used to establish cell suspensions to be cultured as described below without primary callus material in the culture vessel. Callus cultures As described elsewhere (Blaschke, 1977), roughly speaking callus cultures consist mainly of 2 types of cells, i.e. small meristematic cells organized in so called 'meristematic nests' growing with high cell-division activity, and rather large 'parenchyma' cells indicating low cell division rates which connect the meristematic nests with each other. These cells seem to be derived from the meristematic regions. As in other bioassays like carrot tissue cultures (Neumann, 1969; Schafer, Blaschke & Neumann, 1978) adding kinetin to the nutrient medium increases the percentage of meristematic cells whereas in minus kinetin treatments parenchyma cells are more predominant. In elaborate studies C-value distribution in meristematic as well as in parenchyma regions of the primary callus material as influenced by kinetin was followed for a culture period of 21 days. Although not all the data obtained can be given here (see also Blaschke, 1977), the major conclusions derived from these investigations can be seen from Fig. 2 in which the data obtained for haploid callus material are summarized. Independently of kinetin supplement in parenchyma regions a broad scattering of C-values between 1 C and 16 C was found. In meristematic regions, however, C-values are very homogeneous, and particularly in kinetin-supplemented treatments 1 C and 2 C values (exclusively) were found corresponding to G x and G % phase of haploid cells. In minus kinetin treatments generally the same picture emerges, however, 2 C values are more predominant (see also Blaschke et al. 1978) and a negligible number of 3 C and 4 C nuclei were detected. Although no data will be given here, generally speaking the same picture emerges on C-value distribution of diploid callus cultures, although somewhat less pronounced (Blaschke, 1977). High cell-division activity as it occurs in meristematic regions apparently favours the accumulation of cells with the lowest possible C-value of a given ploidy level (see also Blaschke et al. 1978).

4 L Culture -- period: 14 days Parenchyma +Kin. Culture period: 21 days Parenchyma +Kin I Meristematic +Kin. Meristematic +Kin. Parenchyma -Kin. Parenchyma -Kin. Meristematic -Kin Meristematic -Kin. F 3 ~ M Relative units Relative units Fig. 2. Profiles of C-value distributions of meristematic and parenchyma regions of callus cultures (n) derived therefrom at various stages of a 3-week culture period of Datura irmoxia as influenced by kinetin.

5 Cell suspensions Ploidy levels of haploid and diploid Datura cultures 369 In Fig. 3 C-value distribution patterns in cell suspensions developed during culture of primary callus material as described above are given for 2 different periods of culture. The broad scattering of C-values resembles closely the results obtained for parenchyma regions, in fact this free-floating cell material is derived from parenchyma portions from the periphery of callus material due to mechanical impact as it occurs during liquid culture. 1C I I 4C 8C C Relative units Fig. 3. Cytogenetic profile of ' autonomous' callus suspensions of haploid and diploid Datura iimoxia at various stages of culture (+ inositol + IES + kinetin). If this free-floating cell material is separated from the primary calli and subcultured in fresh medium of the same composition as was used for the primary explants, a growth pattern emerges as summarized in Fig. 4. A rather high proportion of cell material < 250 fim is subject to death during the first 2 weeks of culture (and this is more pronounced in originally haploid samples consisting mainly of single cells and small clusters of up to 10 cells). However, from about this date onward cell material < 250 fim, i.e. small clusters, accumulates, reaching a maximum about 1 week later. Apparently in cell clusters of size < 250 fim, active cell division goes on, leading in particular in haploid samples to a steep increase in the number of cells as

6 37 R. Kibler, J. Blaschke, E. Forche and K.-H. Neumann 3 o d 2 10 s, 3- B h fl4 '21 ht y'' * 10 J Time in culture, days Fig. 4. Growth of haploid (A) and diploid (B) cell suspension cultures of Datura innoxia. A, total no. of cells/10 ml nutrient solution; A, cell no. of secondary callus material > 250 fun; 0, no. of'free' cells < 250 /an. shown in Fig. 4. This agrees with C-value distribution patterns in these cell suspensions as given in Fig. 5. Although at t 0 as already shown in Fig. 3 C-values of up to 16 C are found in haploid as well as in diploid samples, after 4 weeks of culture of haploids in clusters > 250 /«n which grow with high cell division activity (see Kibler, 1978) only 1 C and some 2 C values are found. In slowly growing cell material of < 250 /tm, still a broad range of C-values is detected resembling closely the data obtained for

7 Ploidy levels of haploid and diploid Datura cultures 371 S 8 2 For legend, see following page.

8 372 R. Kibler, J. Blaschke, E. Forche and K.-H. Neumann the whole cell suspension at t 0. As already observed in callus material (see above), apparently high cell-division activity opposes the accumulation of cells with high C-values. In fact as an analogy, the cell clusters resemble very closely the meristematic nests in callus material as described above and the cellular fraction < 250 /im possibly could be compared to the cells of parenchyma tissue of cultured explants. This is also indicated by protein-distribution patterns obtained by microdisk electrophoresis (Blaschke, 1977). However, using cell suspensions of diploid origin, cell division in clusters larger than 250 /tm is less than in haploid samples and this is paralleled by less homogeneous C-values in these cultures. CONCLUSIONS In an earlier paper (Blaschke et al. 1978) it was shown that under comparable conditions cell-cycle duration of haploid cells is shorter than that of diploids. In addition to this, a supplement of kinetin shortens cell-cycle duration even further. In the present paper results are presented which indicate a close correlation between cell division activity and C-value homogeneity which results in the accumulation of cells with the lowest possible C-value of a given ploidy level. Putting both sets of data together, apparently due to the shorter cell cycle duration of cells of lower ploidy level in actively dividing cell material in callus cultures, as well as in cell suspensions, an accumulation of haploid cells in material of n origin and diploids of in origin takes place. This tendency can be further accentuated by a kinetin supplement to the culture medium. This accumulative effect on haploid and diploid cells respectively is even more pronounced in cell suspensions than in callus cultures, which may be due to a high rate of decay of free-floating single cells originating from the periphery of actively dividing callus cultures and cell clusters where usually high C-values are found (Kibler, 1978). The results reported in the present paper were applied to develop a procedure to obtain suspensions consisting of ploidyhomogeneous cells suited for experiments studying cell cycle synchronization as published elsewhere (Blaschke et al. 1978). Fig. 5. C-value profiles of haploid (A) and diploid (B) Datura innoxia cell suspension cultures: at the beginning of the experiment (* ) (top); and after a 4-week culture period (tig), for secondary callus material, and for 'free cells' (middle and lower histograms, respectively). Horizontal axis indicates C values and relative fluorescence units, in % of total nuclei measured. NL + inositol + IES +kinetin, C = 13-5 relative units.

9 Ploidy levels of haploid and diploid Datura cultures 373 REFERENCES BLASCHKE, J. R. (1977). Histologische, cytochemische und biochemische Untersuchungen zur Charakterisierung des Kinetineinflusses auf die Entwicklung haploider und diploider Kallu8kulturen von Datura innoxia Mill. Dissertation Giessen. BLASCHKE, J. R., FORCHE, E. & NEUMANN, K.-H. (1977). Standardisierung mikrofluorometrischer Bestimmungen des Ploidiegrades pflanzlicher Zellen. Z. PflZUcht. 78, BLASCHKE, J. R., FORCHE, E. & NEUMANN, K..-H. (1978). Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill, and its synchronization. Planta 144, FORCHE, E. & NEUMANN, K.-H. (1977). Der Einfluss verschiedener Kulturfaktoren auf die Gewinnung haploider Pflanzen aus Antheren von Datura innoxia und Nicotiana tabaccum ssp. Z. PflZUcht. 79, KIBLER, R. (1978). Untersuchungen zum Einfluss des Ploidieniveaus auf Wachstum, Differenzierung und Alkaloidsyntheseleistung von Datura innoxia Zellsuspensionskulturen. Dissertation Giessen. KIBLER, R., FORCHE, E. & NEUMANN, K.-H. (1979). The influence of kinetin on cytogenetic stability of haploid cell suspension cultures of Datura innoxia Mill, (in preparation). NEUMANN, K.-H. (1965). In Les Phytohormones et V Organogenise, vol. 38, p. 95, Les Congres et Colloques de l'universite de Liege. NEUMANN, K.-H. (1969). Mikroskopische Untersuchungen zum Wachstumsverlauf von Explantaten verschiedener Gewebepartien der Karottenwurzel in Gewebekultur. Mikroskopie 25, NEUMANN, K.-H., BLASCHKE, J. R., FORCHE, E. & KIBLER, R. (1978). 4th int. Congr. PI. Tissue and Cell Cult., Book of Abstracts, p Calgary: University of Manitoba Press. SCHXFER, A., BLASCHKE, J. R. & NEUMANN, K.-H. (1978). On DNA metabolism of carrot tissue cultures. Planta 139, SUNDERLAND, N. (i973). Nuclear cytology. In Plant Tissue and Cell Culture (ed. H. E. Street). Oxford, London, Edinburgh, Melbourne: Blackwell. VANZULLI, L., MAGNIEN, E. & OLIVI, L. (1980). Caryological stability of Datura innoxia calli analysed by cytophotometry for 22 hormonal combinations. PI. Sci. Letters (in press). (Received 26 October Revised 25 January 1980)

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