Plant Cell Reports (1997) 16: 884–887
© Springer-Verlag 1997
A. Rival · T. Beule · P. Barre · S. Hamon · Y. Duval M. Noirot
Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants
Received: 17 February 1997 / Revision received: 13 May 1997 / Accepted: 22 May 1997
Abstract Flow cytometric analysis performed on two different crosses of dura ×pisifera oil palm gave an accurate estimation of nuclear DNA content. The genome size of Elaeis guineensis was found to be 2C=3.76±0.09 pg and therefore ca. 3.4×109 bp. Embryogenic calli and plants showed the same ploidy level, but the measured 2C DNA values differed significantly. No variation in the ploidy level between three different types of calli originating from foliar explants, namely nodular compact callus, fast-growing callus and friable callus was observed. Since fast-growing callus (FGC), already identified as a source of ‘mantled’ phenotype variants, did not show any difference in their ploidy level, these results are consistent with the hypothesis of an epigenetic origin for this type of somaclonal variant. Key words Micropropagation · Ploidy · Somaclonal variation · Somatic embryogenesis Abbreviations AVP Acclimatised vitroplant · FC Friable callus · FGC Fast-growing Callus · IRP In-vitro rooted plantlet · NCC Nodular compact callus · SDP Seedderived Palm
Introduction
Oil palm in vitro micropropagation through somatic embryogenesis is now used on a large scale. Nevertheless, a proportion of somatic-embryo-derived oil palms has been found to display the ‘mantled’ somaclonal variant phenotype, which affects the structure of flowers of both sexes (Corley et al. 1986). The phenotypic fidelity of regener-
ants depends on the type of callus used for micropropagation. Clonal oil palm plantlets originating from nodular compact callus (NCC) have been shown to exhibit the ‘mantled’ variant phenotype at an average level of 5%, whereas this rate has been found to attain 100% with plantlets derived from fast growing callus (FGC). A third type of callus, named friable callus (FC) has been used as the starting material for the initiation of embryogenic suspension cultures (de Touchet et al. 1991); its impact on genetic fidelity is currently under investigation (Duval et al. 1995). Several potential markers of the oil palm ‘mantled’ abnormality, such as polypeptide patterns (Marmey et al. 1991) and endogenous cytokinins (Besse et al. 1992), have been investigated, but difficulties have been encountered when using these procedures on a large scale. Our attention is now focusing on the cytometric approach, in order to investigate the potential effect of somaclonal variation on genome size. Indeed, genome size can be altered by in vitro culture through changes either in the chromosome number (Orton 1980; Karp et al. 1987) or in the ploidy level (Lee and Phillips 1988). Flow cytometry is a rapid and efficient method to estimate nuclear DNA content (Galbraith et al. 1983). This technique has proven suitable for routine large-scale studies of ploidy level. Flow cytometric studies of the nuclear DNA content of Elaeis guineensis have not, however, been described to date. The flow cytometry method was applied in the work described here to (1) compare the ploidy level in in vitro cultures and in seed-derived adult oil palms and (2) detect putative variations in DNA content between NCC, FGC and FC.
Communicated by N. Amrhein A. Rival (½) · T. Beule · P. Barre · S. Hamon · Y. Duval M. Noirot CIRAD-CP/ORSTOM · Laboratoire de Ressources Génétiques et Amélioration des Plantes Tropicales, BP 5045, F-34032 Montpellier, Cedex 01, France Fax no.: +33-467-54-78-00 E-mail:
[email protected]
Materials and methods Plant material Seed-derived adult oil palms (SDPs) were obtained from hybrid dura×pisifera commercial crosses G6446 [(D8D×D8D)×(L9T×L13T)] and D1439 [(D10D×D10D)×PO1097P]. Acclimatised vitroplants
885 (AVP) were obtained through somatic embryogenesis as described previously (Duval et al. 1995) and analysed after a 2-year cultivation period in a computer-monitored greenhouse (27±2°C, relative humidity=70±5%, PAR=150–250 µmol m–2 s–1). In-vitro-rooted plantlets (IRPs) were sampled at the end of the rhizogenesis phase, immediately before transplanting to ex vitro conditions. In each of the latter three cases (SDP, AVP and IRP), estimation of nuclear DNA content was carried out on leaf tissue. All the analysed calli (FGC, NCC and FC) were obtained through in vitro culture of foliar explants according to Pannetier et al. (1981) and de Touchet et al. (1991). Sample preparation
between the six types of plant material was performed from a total of 57 extractions distributed as follows: 13, 10, 4, 10, 10 and 10 for NCC, FGC, FC, IRP, AVP and SDP respectively. One-way ANOVA was applied to test genotypic differences within SDPs, and to test variations between the six types of plant material. Multiple mean comparison was performed using the test of Newman (1939) and Keuls (1952).
Results and discussion
Nuclei were extracted from callus cultures and plant leaves by chopping in the lysis buffer of Dolezel et al. (1989), slightly modified in its composition (0.5% Triton X-100 and pH 9.2). Leaves were chopped in a Petri dish in the lysis buffer. The solution was then filtered through a nylon cloth (50 µm mesh size).
Nuclear DNA content did not differ between crosses D1439 and G6446 (F1,17=0.51; P=0.49). This allows the grouping of all the data corresponding to SDPs for further analysis.
Staining of nuclei
Ploidy level in calli and plants
Each sample contained about 0.3 ml of filtrate. Nuclei were stained with propidium iodide (Sigma P4170). The saturating staining concentration used was 330 µg ml–1 according to Barre et al. (1996). After a staining time of 3 min, the filtrate was vigorously shaken in order to separate nuclei clusters.
The most noteworthy observation is that calli and plants, whether derived from seeds or in vitro somatic embryogenesis, showed the same ploidy level (Table 1). The presence of one single peak in all the analysed samples (Figs. 1, 2) demonstrated the presence of homogenous 2n nuclei, without any detectable aneuploidy. Our results confirm
Cytometric measurements A FACScan cytometer (Becton Dickinson) was used with an argon laser (15 mW) at 488 nm. FL2 area and FL2 width were collected over 1024 channels. Measurements were performed on FL2 area histograms. At least 500 nuclei per sample were counted. The zero offset of the analog-to-digital converter was checked with Fluoresbrite calibration grade 2-µm beads (Polysciences) as described by Barre et al. (1996). Beads were used as an external standard and checked every four samples. Estimation of nuclear DNA content was made using nuclei from Petunia hybrida (2C=2.85 pg) as a reference (Marie and Brown 1993). The high voltage was set at 500 V and remained constant throughout the experiment. Other technical parameters have been previously described (Barre et al. 1996). Experimental design The oil palm genome size was determined on two plants (SDP) derived from different crosses (five extractions per plant). Comparison
Fig. 1 Histogram of fluorescence intensities in oil palm nuclei isolated from leaves from seed-derived palms and stained with propidium iodide. The fluorescence pulse area was collected on a 1024-channel analyser
Table 1 Flow cytometric determination of nuclear DNA content in various lines of oil palm (Elaeis guineensis Jacq) callus cultures, somatic-embryo- and seed-derived plants. Results of one-way ANOVA: F5,51=7.74 and P=0.00002. Means followed by the same letter are not significantly different as determined by the Newman and Keuls’ test Plant material
Nuclear DNA content (pg)
Seed-derived palms Acclimatised vitroplants In-vitro-rooted plantlets Fast-growing callus Nodular compact callus Friable callus
3.786 ±0.125 b 3.701 ±0.223 b 3.790 ±0.164 b 3.295 ±0.379 a 3.290 ±0.432 a 3.212 ±0.223 a
886 Fig. 2 Histogram of fluorescence intensities in oil palm nuclei isolated from nodular compact callus and stained with propidium iodide. The fluorescence pulse area was collected on a 1024-channel analyser
those obtained by Jones et al. (1982), who clearly showed the presence of only diploid cells in oil palm plantlets regenerated from tissue culture. These authors demonstrated that deviations from cytological normality in calli and suspension cultures occurred, but it was presumed that these abnormal cells did not contribute to embryogenesis. In our experiment, flow cytometry did not reveal such abnormalities in callus cultures. Kubalokava et al. (1996) reported the occurrence of polyploid and mixoploid cells during the regeneration of Cucumis sativus from embryogenic calli. Conversely, Libiakova et al. (1995) noted that both longterm callus cultures and regenerated shoots of hybrid Abies remained at the diploid level during tissue culture. Konan et al. (1994) obtained the same type of results when analysing the ploidy level of somatic-embryo-derived plantlets of Manihot esculenta by flow cytometry.
2C value for oil palm Flow cytometric estimation of the nuclear DNA content of oil palm (2C=3.76±0.09 pg) was higher than the estimation by the Feulgen microdensitometric method (Jones et al. 1982), which was 2C=2.4±0.4 pg (standard=mouse lymphocytes), or 2C=1.98±0.3 pg (standard=chicken erythrocytes). The Feulgen microdensitometry method has been compared to the flow cytometry method in a number of cases and, generally a good correlation has been found between the two (Michaelson et al. 1991). For oil palm, the discrepancies are quite large and might therefore be attributable not only to the differences between the techniques, but also to the type of standard used for estimation. Genome size and somaclonal variation
Fluorochrome accessibility in plants and calli The relative fluorescence of nuclei was found to be significantly higher in leaves than in calli (3.76 versus 3.27 pg/nucleus, respectively), the difference corresponding to 14% (Table 1; Figs. 1, 2). The six types of plant material analysed could be subdivided into two groups: calli versus others. These discrepancies in estimated DNA contents between calli and plants could be attributed to differences in chromatin condensation. Indeed, chromatin condensation is very sensitive to small fluctuations in the nuclear environment (Darzynkiewitz et al. 1975; Johnston et al. 1996) such as changes in bivalent cations, in pH or in light. In humans and animals as in plants, the degree of chromatin condensation has been shown to vary between tissues (Dolezel et al. 1989; Vago 1993; our submitted results).
The three types of calli (NCC, FGC and FC) did not differ in their nuclear DNA content (Table 1). As FGC has been described as a source of somaclonal variants, the ‘mantled’ variation is not associated with variations in the nuclear DNA amount in embryogenic callus lines. This result thus reinforces the hypothesis of an epigenetic origin for somaclonal variation in oil palm. Acknowledgements Thanks are due to the directors of INRABSRPH in Pobé, Benin and IRA La Dibamba in Cameroon for the generous supply of selected germinated oil palm seeds. Miss C. Recalt is thanked for her excellent glasshouse management. We are grateful to Dr. J. Tregear for English corrections.
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