faba (4C = 53.31 pg) (Bennett and Smith ... the same 2C (G,) interphase nucleus, after selecting different .... scanned in a Vernon PHl-type densitome- ter, and ...
Variation of Repetitive DNA Sequences in Progenies of Regenerated Plants of Pisum sativum A. Cavallini, L. Natali, E. Polizzi, and T. Giordan!
From the Department of Agricultural Plant Biology ol the University, Genetics Section, Via Matteottl 1/B, I56124 Pisa, Italy. Research reported herein was supported by the National Research Council of Italy, Special Project RA1SA, subproject no. 2, paper no. 2475. Address reprint requests to Dr. Cavallini at the address above. Journal of Heredity 1996:87:233-237; 0022-1503/96/S5.00
Genome size variations have often been related to environmental stimuli and can be produced by stress conditions occurring during plant development (Cullis 1990; Schneeberger and Cullis 1991; Walbot and Cullis 1985). During recent years, quantitative DNA variations have often been described in plants regenerated through in vitro tissue culture. It is known, in fact, that in vitro culture may determine cryptic DNA alterations as amplification, underreplication, or underrepresentation of certain DNA sequences or families (Cecchini et al. 1992; Nagl 1990): these alterations might produce some of the so-called somaclonal variants (Brown and Lorz 1986; Karp 1991). Variations in genome size through differential DNA replication during in vitro culture were first reported in regenerated doubled haploid of Nicotiana spp. (De Paepe et al. 1982; Dhillon et al. 1983). Concerning the DNA sequences involved in the variations, differential DNA replication only rarely affects functional genes in plants, apart from ribosomal DNA (Brown et al. 1991; Caretto et al. 1994; Donn et al. 1984; Xiao et al. 1987); usually, nuclear DNA content variation in regenerated plants is mainly related to changes in the frequency of repetitive, noncoding DNA (Caboche and Lark 1981; Deumling and Clermont 1989; Karp et al. 1992; Kidwell
and Osborn 1993; Lapitan et al. 1988; Natali et al. 1995). However, these phenomena are usually studied in the regenerated plants, and little is known on genome size variation in the subsequent generations. We have analyzed differential DNA replication in Pisum sativum plants (belonging to the experimental line "5075") regenerated from 1-year-old calli and observed a 7-15% reduction in 4C DNA content, as determined by Feulgen cytophotometry: both repeated (including ribosomal DNA) and "unique" (as isolated kinetically) sequences were found to vary their frequency (Cecchini et al. 1992). On the contrary, no quantitative variations were established for DNA sequences coding for storage proteins, as legumin and lectin (Bernardi et al. 1995). Two hypotheses may explain the observed variations in "unique" sequences of pea regenerated plants (Bernard! et al. 1995): (a) they might represent repeated gene elements (often originating from transposons) undergoing mutations during evolution (Smyth 1991), or (b) they might be interspersed within repetitive DNA and change concomitantly (Brown et al. 1991). Because regenerated pea plants do not usually show any gross phenotypic alterations, it may be presumed that changes in copy number are probably limited to uncoding and/or repeated DNA se-
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The frequency variation of highly repeated (HR) DNA sequences was studied in plants regenerated through in vitro culture of macerated vegetative apices of Pisum sativum and their progenies. Feulgen cytophotometry showed that regenerated plants (R,) have 6-7% more DNA per nucleus than control plants; this difference is maintained in the subsequent generation (RJ. Slot-blot analyses using isolated highly repeated sequences as probe indicated that an increased frequency of these sequences occurs in regenerated plants and their progenies. These results were confirmed by a series of experiments: (a) metaphase chromosomes were longer in R2 than in control plants; (b) cytophotometric analyses of chromatin structure after Feulgen-staining showed that condensed chromatin is more represented In R, than In control plants; and (c) thermal denaturation of isolated HR sequences indicated that a new HR family appears in R, and is maintained in R2 plants. These results suggest that, in this species, the DNA extrasynthesized during in vitro culture is, at least In part, Integrated in the chromosomes and transmitted to the progeny.
quences or to genes that do not have major effects. To determine whether genome size variations are transmissible to subsequent generations, through meiosis, we studied the selfed progenies of regenerated plants of another pea experimental line. This line showed, in regenerated plants, a 6-7% increase of genome size. In this article, we report cytophotometric, biochemical, and molecular analyses on the behavior of highly repeated sequences in these plants. Materials and Methods
Cytophotometric Analyses Cytophotometric analyses were performed after Feulgen staining of slides, according to standard procedures (Cavallini et al. 1981). Root apices and young leaflets from control plants (C), regenerated plants (RJ, and their progenies (Rj) were fixed in ethanol/acetic acid 3:1 (v/v) for 24 h and then treated in 4% pectinase (Sigma) for 20-30 min at 37°C. Squashes were made in a drop of 45% acetic acid, and cover slips were removed by the dry ice method (Conger and Fairchild 1953). Slides were then hydrolyzed in HC1 at different conditions: either 1 N at 60°C for 8 min or 5 N at 20°C for 10-60 min. Optimal
234 The Journal of Heredity 1996 87(3)
On the same slides, using a Leitz MPV3 integrating microdensitometer, the Feulgen/DNA absorption of chromatin fractions with differing condensation were determined by measurements on one and the same 2C (G,) interphase nucleus, after selecting different thresholds of optical density in the instrument (Cavallini et al. 1989). The instrument reads all parts of the nucleus where optical density is greater than the preselected limit: at low thresholds all the chromatin is measured, whereas at growing thresholds only more condensed chromatin is read. The first derivative curve (in absolute values) of the absorption profiles allows discrimination among differently condensed chromatin fractions. All absorption values were normalized (absorption = 100 at minimum optical density threshold) to facilitate comparisons. For metaphase length analysis, C and R2 seeds were germinated and root tips collected at the same time. Root tips were treated with 0.02% colchicine (Sigma) for 4 h, fixed as above, hydrolyzed for 8 min in N HC1 at 60°C, Feulgen-stained for 1 h, and squashed in a drop of 45% acetic acid. Slides were then made permanent as described above and analyzed. DNA Extraction and Fractionatlon DNA was purified according to the method devised by Doyle and Doyle (1989) with
modifications. Leaves from individual C, R,, and R2 plantlets were ground in a mortar in CTAB isolation buffer (1.5% (w/v) CTAB (Sigma), 1.4 M NaCl, 0.2% (v/v) 2mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCI, pH 8.0) at 60°C. Samples were incubated at 60°C for 30 min with occasional gentle swirling and then extracted once with chloroform/isoamyl alcohol (24:1, v/ v). After centrifugation (3,000 X g) at room temperature, nucleic acids were precipitated from the aqueous phase by adding 2/3 volumes of cold isopropanol and then spooled with a glass hook, washed in 76% ethanol, 10 mM ammonium acetate for 1-2 h, allowed to dry briefly, and resuspended in water. For further purification, solid CsCl and ethidium bromide were added to the nucleic acids solution up to final concentrations of 0.8 mg/ml and 200 ^g/ml, respectively. The solution was centrifuged at 175,000 X g in a Beckman L5-65 ultracentrifuge for 48 h using the 65 Ti rotor and the DNA band, visualized under long-wave UV illumination, was collected. Ethidium bromide was then removed by gentle inversion of the solution with n-butanol and dialyzed against water at 4°C for 3 h. Finally, DNA was ethanol-precipitated and resuspended in the appropriate buffer. For fractionating DNA at 10° Cot value, it was solubilized in 0.12 M Na-phosphate buffer pH 7.0 and sheared by sonication in an MSE sonicator at medium energy output for 5 X 5" with 10" intervals at 4°C. The DNA was then denatured for 10 min at 103°C and allowed to reassociate according to Britten et al. (1974) up to Cot = 10° (Murray et al. 1978) in order to isolate highly repeated sequences (HR). DNA was fractionated by elution through a hydroxylapatite column equilibrated in the same buffer as above: single-strand DNA was eluted with the same buffer, and reassociated sequences were recovered by elution with 0.5 M phosphate buffer. Biochemical Analyses Thermal denaturation of reassociated HR DNA sequences from C, R,, and R2 plantlets were performed in 0.12 M Na-phosphate buffer using a Shimadzu UV 2101 PC spectrophotometer equipped with a temperature programme controller. Increase in hyperchromicity at 253 nm was continuously recorded at 65-100°C. First derivative curves of denaturation profiles allowed different DNA families to be distinguished according to their melting temperatures (Turner et al. 1993).
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In vitro Culture In vitro culture of vegetative pea shoots was performed on the experimental line "E," according to Natali and Cavallini (1987). Vegetative apices were excised from sterile germinating seeds, mechanically macerated, and placed on solid MS (Murashige and Skoog 1962) medium supplemented with 3% sucrose, 0.5 mg/1 6benzylaminopurine, and 0.2 mg/1 naphtalene-acetic acid (pH 5.7) and maintained under white fluorescent light of 2,000 lux, using a light and dark cycle of 16/8 h. Shoot regeneration from calli started after 1 month of culture. Shoots regenerated from different calli after 1 year of culture were excised, cultured on the same medium to achieve plant development, and then transferred to half-strength MS containing 2 mg/1 indolebutyric acid for root induction. Rooted plantlets were transplanted into pots containing soil and sand, and covered with plastic bags to prevent desiccation during the first 2 weeks. Regenerated plants were then cultivated in the greenhouse up to flowering and producing seeds (Rj). Control and R2 seeds were germinated and cultivated in the greenhouse in the same conditions as the R, plants.
hydrolysis times were determined previously (Cavallini and Natali 1990) for hot hydrolysis and before these experiments for cold hydrolysis. After hydrolysis, slides were Feulgen-stalned in 0.5% basic fuchsin for 1 h at room temperature, washed twice in SO2 water (0.09 M sodium metabisulphite, 0.16 N HC1) for 15 min, dehydrated, and mounted in D.P.X. balsam (BDH Chemicals). Nuclear DNA content was estimated by a Barr and Stroud, GN5type, integrating cytophotometer at the wavelength of 550 nm. Slides to be directly compared were concurrently stained; when simultaneous processing was not possible, due to the large number of preparations to be analysed, squashes made with root tips of a single plantlet of Vicia faba (4C = 53.31 pg) (Bennett and Smith 1976) were concurrently stained for each group of slides and used as control for the experimental technique: only when 4C DNA value of standard Vicia faba (in arbitrary units) was the same, then the concurrently stained pea slides of different experiments were compared. 4C-values of standard Vicia faba were also used to convert 4C DNA values of pea plants to picograms.
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Figure 1. Diagram representing frequency variation of HR sequences In single control plants (C), In plants regenerated after 30-60 days of culture (RJ, and In their progenies (RJ. Single plant values were obtained by densltometrlc analyses of slot blots probed with HR sequences and were normalized to facilitate comparisons. Black bars represent the mean, the white boxes show the distribution of single values, and external bars are the fiduciary limits (P ^ .001).
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