In vitro culture of Aloe barbadensis Mill.: quantitative DNA variations in regenerated plants

Plant Science, 91 (1993) 223-229 Elsevier Scientific Publishers Ireland Ltd.

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In vitro culture of Aloe barbadensis Mill." quantitative DNA variations in regenerated plants A. Cavallini a, L. N a t a l i a, G. Cionini b, O. Sassoli a a n d I. C a s t o r e n a - S a n c h e z c aDipartimento di Biologia delle Piante Agrarie dell'Universith, blstituto di Mutagem,si e Differenziamento, Pisa (Italy) and "Centro de lnvestigacion Cientifica de Yucatan. Merida ( Mex&o) (Received December 31st, 1992; revision received March 8th, 1993; accepted March 30th, 1993)

Six plants of Aloe barbadensis regenerated from callus were studied as far as chromosome number: five plants were diploid and one tetraploid. Cytophotometric analyses of Feulgen-stained early-prophase nuclei showed a 22.5% basic DNA content variation among diploid plants. Both increases and diminutions of DNA were recorded, rclated to precise chromatin fractions (either euchromatin and heterochromatin), as distinguished by their condensation in interphase nuclei. Variations were also found among regenerated plants related to the amount of ribosomal DNA. As far as the phenotypic effect of these variations, analysis of epidermal cell area showed that nuclear DNA content affects positively cell dimensions.

Key words: Aloe barbaderL~is Mill.; basic DNA content variation; cell dimensions; cytophotometry; heterochromatin; regenerated plants

Introduction

It is known that, in many cases, plants regenerated through tissue culture differ from the parental type due to somatic nuclear mutations [1]. The mutational events may depend on the plant species, the genotypes involved, the type of explant, and the culture media and condition [2]; they may involve genomic, chromosomal and gene mutations [3], the activation of transposable elements [4], changes in methylation patterns [5]. While karyological changes (i.e. polypioidy, aneuploidy, chromosomal mosaicism) have been amply documented [3], more cryptic DNA alterations as amplification or loss of DNA and re-arrangements of nuclear DNA have only recently been observed Correspondence to: Prof. A. Cavallini, Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Via Matteotti I/B, 1-56124 Pisa, Italy. Abbreviatiora': CTAB, hexadecyltrimethylammonium bromide; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS, Murashigc and Skoog medium.

in tissue culture derived plants ([6-8] and other references therein). Aloe barbadensis Mill. (A. vera L.) is cultivated for the extraction of the juice, widely used today in pharmacy as well as in cosmetics [91. Aloe plants are normally vegetatively propagated but, recently, in vitro culture technologies have been employed to accelerate plant production via shoot micropropagation [10,11]. Moreover, since vegetatively propagated plants are genetically uniform, plant regeneration from callus has been tested to generate new variability. In a previous paper [12] we described a protocol for plant regeneration from leaf base explant derived calli: though only 1-2% explants formed regenerating calli, these regenerated many shoots, probably by organogenesis, that were cultivated in the greenhouse. These shoots did not generally show abnormal chromosomal numbers [12]. Therefore we have looked for more cryptic genetic changes: in the present paper we report the results of this study.

224 Materials and Methods

Plant materhTl and in vitro culture Regenerated plants of A. barbadensis were achieved according to Castorena-Sanchez et al. [12]. Leaf bases were explanted from a single plant, surface sterilized and cultured in MS [13] containing 2,4-D 0.25 mg/l, kinetin 1 mg/l, paminobenzoic acid 0.1 mg/l and tyrosine 100 mg/l. After 30-45 days, compact calli were transferred to MS containing 2,4-D 0.025 mg/l plus 6-benzylaminopurine 1 mg/l. Regenerated shoots were transferred to hormone-free MS for rooting and then to earthen pots in the greenhouse where they grew for 3 years before being analyzed. This study reports the results of six regenerated plants, compared to the plant from which explants were taken. Cytological and cytophotometric analyses Cytological analyses were made on both root apices and leaflet meristems, collected and fixed in ethanol/acetic acid (3:1, v/v). For chromosome counts the materials were treated with a 0.52/0 solution of colchicine (Sigma) for 4 h prior to fixation, then Feulgen-stained (after hydrolyses in N HC1 at 60°C for 8 min), dehydrated and mounted. Cytophotometric analyses were performed according to the conditions established for Aloe [14]. Fixed samples (not treated with colchicine) were put in a 5% solution of pectinase (Sigma) for 1 h at 37°C and then squashed in a drop of 45% acetic acid. After removing the cover-slips, the preparations were hydrolyzed in N HCI at 60°C for 8 min, Feulgen-stained (in a 0.5% basic fuchsin solution) for 1 h, and subjected to three 10-min washes in SO 2 water, prior to dehydration and mounting in DPX (BDH). Slides that were to be directly compared were always stained simultaneously. Feulgen DNA absorptions in individual cell nuclei were measured at the wavelength of 550 nm using the Barr & Stroud integrating microdensitometer, type GN5. With the same instrument, and at the same wavelength, the Feulgen/DNA absorption of chromatin fractions with differing condensation were determined by measurements on one and the same 4C (G2) interphase nucleus, after selecting different thresholds of optical density in the instru-

ment [15]. The instrument does not read all parts of the nucleus where optical density is greater than the preselected limit, regarding them as a clear field: at low thresholds only euchromatin is read, while, at high thresholds all the nucleus (euchromatin plus heterochromatin) is measured. The first derivative curve of the absorption at different thresholds allows to discriminate among differently condensed chromatin fractions. For each plant, 10 nuclei x 3 slides × 5 roots were measured. DNA extraction DNA was extracted from fresh leaf tissues according to the method devised by Doyle and Doyle [16] with minor modifications. Central leaf portions were ground in a preheated mortar in CTAB isolation buffer (3% (w/v) CTAB (Sigma), 1.4 M NaCI, 0.2% (v/v) 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, (pH 8.0)) at 60°C. The samples were incubated at 60°C for 30 rain with occasional gentle swirling and then extracted once with chloroform/isoamyl alcohol (24:1, v/v). After centrifugation (5000 rev./min) at room temperature, nucleic acids were precipitated from the aqueous phase by adding 2/3 volumes of cold isopropanol. Then nucleic acids were spooled with a glass hook, washed in 76% (v/v) ethanol, 10 mM ammonium acetate for 1-2 h, allowed to dry briefly and resuspended in TE buffer (TE: 10 mM Tris-HCl, 1 mM EDTA, pH 7.4). DNA blotting For slot DNA blots, 1 ~g DNA samples were denatured by heating at 37°C for 10 min in 0.5 M NaOH then neutralized by addition of an equal volume of 2 M ammonium acetate. Scalar dilutions of DNA from 0.5 to 0.03125 t~g were loaded on nylon filters (Hybond-N; Amersham) using a commercial slot blotting apparatus (Minifold II; Schleicher & Schuell). An EcoRI 18+25S rDNA repeat of Phaseolus coccineus cloned into Lambda EMBL4 arms and subcloned into pUC13 vectors [17] was labeled with digoxigenin I I-dUTP by a DIG-DNA labeling kit (Boehringer) and used as probe. Hybridization was performed according to [18]. Filters were washed sequentially in 2 x , 1 x, 0.3x SSC (SSC: 150 mM NaC1, 15 mM trisodium

225

citrate, pH 7.0) containing 0.05% SDS at 65°C, and hybridization was detected by a DIG-DNA detection kit (Boehringer). Filters were then scanned in a Vernon PHl-type densitometer, and the tracings were used for quantitative determinations. All hybridization experiments were repeated at least six times. As a control, all the filters were rehybridized with PstI maize alcohol dehydrogenase cDNA cloned into pBR 322 vector [19] and labelled with a [32p]dCTP Random Primer kit (Amersham). Filters were then autoradiographed and scanned as above.

Morphometric analyses Epidermal peels were made from the central portion of both the upper and lower faces of adult leaves (30 cm long), fixed in ethanol/acetic acid (3:1) and treated with a 1.2% pectinase solution for 3-4 h at 37°C. Peels were put onto slides and stained with a 2% Giemsa solution. After photocopying photographs on Wattman 3 M paper, epidermal cell area was determined by cutting and weighing cell shapes (100 cells/plant). Results

Karyological analyses in root and leaf meristems showed that all the plants analyzed were diploid (2n = 14) except plant A2, that revealed tetraploid (Table I). Tetraploidy of plant A2 was ascertained also by cytophototometric analyses of parenchyma cells in different leaves (not reported).

Cytophotometric measurements of early prophases in root apices indicated a large variability of basic (4C) DNA content of regenerated plants in respect to the control plant: indeed, when considering only the diploid plants, a 22.5% variation is found between plants A1 and A4 (Table 1). The same Feulgen-DNA absorption values were obtained when analyzing leaf meristem (data not reported). In another cytophotometric analysis the chromatin structure of interphase 4C (G2) nuclei was considered, lnterphase nuclei were measured at different thresholds of optical density and the frequency of differently condensed chromatin fractions was calculated by first derivative curves of absorption (see Materials and methods): values at 0-3 thresholds should indicate the euchromatin, while values at 3-8 thresholds should indicate differently condensed heterochromatin. Optical density profiles of the six plants are reported in Fig. 1: differences may be observed among regenerated plants and in respect of the same control plant. On the basis of Fig. 1, the percentage of heterochromatin (3-8 thresholds) and the ratio between heterochromatin and euchromatin were calculated (Table I). The ratio between heterochromatin and euchromatin show a great variation among plants; however, since no positive correlation was established between heterochromatin content and total nuclear DNA content of the six plants, it may be argued that the quantitative variations observed are not due only to

Table I. Chromosome number, Feulgen absorption of early prophases (4C, a.u.; mean ± S.E.), 1 C DNA content (pg), heterochromatin content and heterochromatin/euchromatin ratio (calculated analyzing optical density of interphase nuclei) of six regenerated and one control plant of A. barbadensis. In the last column, the mean epidermal cell areas in the upper face of adult leaves are also reported Plant

Chromosome number

4C DNA content (a.u.) ± S.E.

DNA pg per haploid ( I C) nucleus

Heterochromatin (%)

itetero-/ Euchromatin ratio

Mean epidermal leaf cell area (a.u.; +S.E.)

AI A2 A3 A4 A5 A6 Control

14 28 14 14 14 14 14

19.88 44.16 23.09 25.65 22.56 23.34 22.54

12.72 28.26 14.78 16.42 14.44 14.94 14.43

80.02 64.97 55.93 43.96 30.46 35.00 54.46

4.01 1.85 1.27 0.78 0.44 0.54 1.20

3501 39(18 3950 4921 3559 4440 3570

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heterochromatin but also to differently condensed chromatin fractions. In another experiment, DNAs were extracted and slot-blot DNA hybridization analysis performed using ribosomal DNA sequences. The densitometric values (each repeated six times) of the slot-blot experiments were normalized (control = 100), averaged (Fig. 2, left) and referred to the basic DNA content of the plant (Fig. 2, right): here

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also, variations in the relative frequency of rDNA sequences are found. Finally, morphometric analyses on leaf epidermal cell area also revealed variations among plants (Fig. 3). The mean epidermal cell areas of the upper face of the adult leaf in the six plants are reported in Table I. No significant variations were ascertained between the upper and lower face of the leaf and among vegetatively propagated plants

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(data not reported). Since in histological section the epidermal thickness did not change among plants, cell area values can directly be correlated to cell volume. If excluding the tetraploid plant, a significant positive correlation (P _< 0.001, Fig. 4) was established between cell area and basic (4C) DNA content (at the diploid level). Discussion

Mutually supporting one another, all the experiments reported in this paper indicate that DNA variability does exist among regenerated plants of

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228 A. barbadensis in respect to the original plant from which explants were taken. Though Feulgen cytophotometry may be subjected to certain reservations [20], it is worth noting that, in our experiments, analogous explants from plants grown in the same conditions were simultaneously stained, measured and compared, i.e., very controlled analytical conditions were established. Moreover, slot DNA blot hybridization and morphometric experiments also confirm the existence of genome variation. Since we analyzed adult (3 years old) plants, it may be supposed that the variations are not a transient response to in vitro culture stress, but they are stable. Nuclear D N A content variation is related to in vitro culture because it does not occur in normal plants: only minor variations were observed as to total DNA (4C = 22.48 + 0.15 a.u.) and heterochromatin content in five vegetatively propagated plants of A. barbadensis developed in the greenhouse [14]. Preliminary data on rDNA content indicate that this sequence is stable among these plants: this parameter is in fact being used in our comparative studies among different Aloe species. Cytogenetic analyses have shown that changes in precise and different chromatin fractions (distinguished by their condensation, Fig. 1) may account for the variation of total DNA content per nucleus observed in prophase nuclei among regenerated plants (Table 1). While in regenerated plants quantitative variations in heterochromatin have been described in literature [8,21-23], variations in euchromatin were found more rarely [7,23]; it is conceivable that both heterochromatic and euchromatin fractions involved in the variations are mainly non-coding sequences, because plants are apparently normal. However, as reported in other species [24-27], changes may also concern house-keeping sequences as ribosomal DNA (Fig. 2), though no correlation was found between rDNA frequency and total DNA content in the regenerants. In this sense, the main fraction of the variable DNA would belong to the so-called 'conformational DNA' [28] that determines general nucleus structure and function. It is also possible that quantitative changes affect nucleotypic characters

[29,30]: for example, a significant correlation was found (in diploid regenerants and the control plant) between basic DNA content and leaf epidermal cell area (Figs. 3 and 4). This relationship was already found in other cases of intraspecific DNA content variation [31-32]. This is an important point, because if the relationship to cell dimension should be confirmed in leaf parenchyma cells (where active principles of aloe drug are accumulated [9]), the basic DNA content might be correlated to the productivity of the plant. Experiments are in progress in this direction. Finally, it is worth noting the presence of a tetraploid plant among regenerants, probably originated from tetraploid cells frequently found in Aloe calli [12]. The regeneration of polyploid plants was reported in other species cultured in vitro [3]. The plant appears normal and indistinguishable among the others, except that it does not produce suckers from the base of the stem. In this plant the epidermal cell area falls within the range of the diploid plants (Table I). Two hypotheses may be made: (i) the epidermal cell nuclei are diploid, i.e., the plant is a diplotetraploid chimera [1]; (ii) the variation of cell area (volume) controlled by the nucleotype is limited within a certain range of DNA content: higher DNA contents have no effect on this parameter. Other comparative studies are in progress on tetraploid and diploid plants. Acknowledgements

Thanks are due to Prof. W.J. Peacock (CSIRO, Division of Plant Industry, Canberra, Australia) and Prof. F. Maggini (Department of Agrobiology and Agrochemistry, University of Tuscia, Viterbo, Italy) for generous gifts of Adh 1 cDNA and cloned rDNA, respectively. Research work supported by National Research Council of Italy, contract no. 93.00161.CT06. References

1 F. D'Amato, Somatic nuclear mutations in vivo and in vitro in higher plants. Caryologia,43 (1990) 191-204. 2 A. Karp, On the current understanding of somaclonal variation. Oxford Surv. Plant Mol. Cell Biol., 7 (1991) 1-58.

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F. D'Amato, Cytogenetics of plant cell and tissue culture and their regenerates. CRC Crit. Rev. Plant Sci., 3 (1985) 73-112. F. Planckaert and V. Walbot, Molecular and genetic characterization of Mu transposable elements in Zea mays: behaviour in callus culture and regenerated plants. Genetics, 123 (1989) 567-578. P.T.H. Brown, DNA methylation in plants and its role in tissue culture. Genome, 31 (1989) 717-729. E. Miiller, P.T.H. Brown, S. Hartke and H. L6rz, DNA variation in tissue culture-derived rice plants. Theor. Appl. Genet., 80 (1990) 673-679. E. Cecchini, L. Natali, A. Cavallini and M. Durante, DNA variations in regenerated plants of pea (Pisum sativum L.). Theor. Appl. Genet., 84 (1992) 874-879. A. Karp, P.G. Owen, S.H. Steele, P.J. Bebeli and P.J. Kaltsikes, Variation in telomeric heterochromatin in somaclones of rye. Genome, 35 (1992) 590-593. A. Cavallini, L. Natali and I. Castorena-Sanchez, Aloe barbadensis Mill. (= A. vera L.), in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, Vol. 15, Springer-Verlag, Berlin, Heidelberg, 1991, pp. 95-106. L. Natali, I. Castorena-Sanchez and A. Cavallini, In vitro culture of Aloe barbadensis Mill.: micropropagation from vegetative meristems. Plant Cell Tiss. Org. Cult., 20 (1990) 71 - 74. H.J. Meyer and J. Van Staden, Rapid in vitro propagation of Aloe barbadensis Mill. Plant Cell Tiss. Org. Cult., 26 (1991) 167-171. I. Castorena-Sanchez, L. Natali and A. Cavallini, In vitro culture of Aloe barbadett~is Mill.: morphogenetic ability and nuclear DNA content. Plant Sci., 55 (1988) 53-59. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant., 15 (1962) 473-497. A. Cavallini, Cytophotometric and biochemical analyses on DNA variations in the genus Aloe L. Int. J. Plant Sci., 154 (1993) 169-174. A. Cavallini and L. Natali, Cytological analyses of in vitro somatic embryogenesis in Brirneura amethystina Salisb. (Liliaceae). Plant Sci., 62 (1989) 255-261. J.J. Doyle and J.L. Doyle, Isolation of plant DNA from fresh tissue. Focus, 12 (1989) 13-15. F. Maggini, G.F. Tucci, A. Demartis, M.T. Gelati and S. Avanzi, Ribosomal RNA genes in Phaseolus coccineus. Plant Mol. Biol. 18 (1992) 1073-1082. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular cloning. A laboratory manual. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982. G.S. Dennis, W.L. Gerlach, A.J. Pryor, J.L. Bennetzen, A. Inglis, D. Llewcllyn, M.M. Sachs, R.J. Ferl and W.J.

Peacock, Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize. Nucl. Acid Res., 12 (1984) 3893-4000. 20 A. Cavallini and L. Natali, lntraspecific variation of nuclear DNA content in plant species. Caryologia, 44 (1991) 93-107. 21 S.S. Dhillon, E.A. Wernsman and J.P. Mikschc, Evaluation of nuclear DNA content and heterochromatin changes in anther-derived dihaploids of tobacco (Nicotiana tabacum) cv. Cocker 139. Can. J. Genet. Cytol., 25 (1983) 169-177. 22 R. De Paepe, D. Prat and T. Huguet, Heritable nuclear DNA changes in doubled haploid plants obtained from pollen culture of Nicotiana sylvestris. Plant Sci. Lett., 28 (1983) 11-28. 23 B. Deumling and L. Clermont, Changes in DNA content and chromosomal size during cell culture and plant regeneration of Scilla siberica: selective chromatin diminution in response to environmental conditions. Chromosoma, 97 (1989) 439-448. 24 J. Landsmann and H. Uhrig, Somaclonal variation in Solanum tuberosum detected at the molecular level. Theor. Appl. Genet., 71 (1985) 500-505. 25 P.T.H. Brown and H. Lrrz. Molecular changes and possible origins of somaclonal variation, in: J. Semal (Ed.), Somaclonal Variations and Crop Improvement, Martinus Nijhoff, Dordrecht, 1986, pp. 148-159. 26 C.A. Cullis and W. Cleary, DNA variation in flax tissue culture. Can. J. Genet. Cytol., 28 (1986) 247-251. 27 R.I.S. BretteU, M.A. Pallotta, J.P. Gustafson and R. Appels, Variation at the Nor loci in triticale derived from tissue culture. Theor. Appl. Genet., 71 (1986) 637-643. 28 W. Nagl, Polyploidy in differentiation and evolution. Int. J. Cell Clon., 8 (1990) 216-223. 29 M.D. Bennett, Nuclear DNA content and minimum generation times in herbaceous plants. Proc. Roy. Soc. Lond., Ser. B, 181 (1972) 109-135. 30 M.D. Bennett, Intraspecific variation in DNA amount and the nucleotypic dimension in plant genetics, in: M. Freeling (Ed.), Plant Genetics, A.R. Liss, New York, 1985, pp. 283-302. 31 L. Natali, A. Cavallini, G. Cionini, O. Sassoli, P.G. Cionini and M. Durante, Nuclear DNA changes within llelianthus annuus L.: changes within single progenies and their relationships with plant development. Theor. Appl. Genet., 85 (1993) 506-512. 32 A. Cavallini, L. Natali, G. Cionini and D. Gennai, Nuclear DNA variability within Pisum sativum (Leguminosae): nucleotypic effects on plant growth. Heredity, 70 (1993) 561-565.