Genome/Génome 42(6): 1134-1143 (1999)
Genome plasticity during the acquisition of embryogenic competence
Chiara Geri, Alessandra Turrini, Lucia Giorgetti, Elisa Nicoletti, and Vittoria Nuti Ronchi

Abstract:
Hypocotyl explants from carrot and other species experience concomitant segregation events and differentiation of homeotic structures during the first 20 days of culture on 2,4-dichlorophenoxyacetic acid (2,4-D). In addition to these cyto-morphological changes, significant amounts of nuclear DNA are lost, the molecular details of which we investigate in this paper. We have developed a slot-blot analysis assay to study the DNA content of a series of carrot samples; besides the leaves, this survey ranged over different culture timepoints: hypocotyls, cell lines, and somatic embryo stages. We carried on to study the relationship between this DNA loss and sequence complexity modulation. Results from probing sequences that correspond to different degrees of complexity, such as medium repetitive and unique sequences as well as sequences belonging to both classes (ribosomal cistrons, ubiquitin, actin, and chalcone synthase), consistently manifested a reduction in DNA levels during the acquisition of embryogenic competence. In some cases, the cultured cells would contain only 10% of the gene copies observed in the reference tissues. Modulation trends also showed that DNA levels of most sequences recover at the torpedo-plantlet stage, which again correlates DNA modulation and the acquisition of embryogenic competence. These results suggest that similar DNA variations may occur in plants in vivo during meiosis, possibly so that meiotic division may be properly completed.

Introduction
Rapid or programmed genomic changes occur regularly in higher plants. It has been suggested that this is evidence of a linkage between genomic structure and the developmental and environmental constraints on the plant life cycle (Walbot and Cullis 1985). However, while amplification events are well documented, DNA loss remains a puzzling phenomenon. Such events are often connected either with endoreduplication or with concurrent amplification of other sequences (Lima de Faria et al. 1975). Most reports focus on these events during senescence within the in vivo context of the plant life cycle. In vitro studies follow a similar vein; few discuss DNA diminution in cultured plant cells or during regeneration, whereas numerous papers are related to DNA amplification.

One of the first reports on this subject concerns the DNA extrasynthesis which precedes cell proliferation during the dedifferentiation process of Nicotiana glauca pith tissue cultured in vitro (Nuti Ronchi et al. 1973; Martini and Nuti Ronchi 1974; Parenti et al. 1973). Selective loss and regain of chromatin in response to environmental conditions has been described during cell culture and plant regeneration of Scilla siberica (Deumling and Clermont 1989) and Pisum sativum (Cecchini et al. 1992).

The cases of DNA modulation so far described in the literature are not in a direct and progressive relationship with the developmental processes, whereas carrot somatic embryogenesis is probably the first case that offers the possibility to observe an emerging relation between embryo development and DNA modulation. In fact, recently we have shown that the process appears coincidental to a meiotic-like segregation mechanism (Nuti Ronchi et al. 1992a, 1992b) and that conferring a gametic-like constitution to cultured carrot hypocotyl cells seems to be a prerequisite for the acquisition of totipotency (Giorgetti et al. 1995; Nuti Ronchi 1995). Moreover, the cultured hypocotyls developed structures resembling floral organs. A tangible DNA loss was shown by microdensitomeric measurements of DNA content per nucleus along the different culture timepoints; a partial recovery was evident at the torpedo and plantlet stages (Nuti Ronchi et al. 1992a, 1992b, Giorgetti et al. 1995, Nuti Ronchi 1995). So far, all these findings, also ascertained in several other plant species (Helianthus, Prunus, Malus; Pitto et al. 1994; Nuti Ronchi et al. 1992c, 1992d; Blando et al. 1991), suggest the hypothesis that the in vitro condition induces, as a primary stress effect, the activation of a reproductive meristem, leading to the expression of floral and reproductive genes.

In this report, we present molecular data showing the occurrence of nuclear DNA modulation during the embryogenic competence acquisition in cultured carrot hypocotyls.

We discuss and interpret our results assuming that the chromosome segregation events and the modulation of DNA sequences are related processes that allow the acquisition of cell totipotency. Furthermore, we continue by hypothesizing that similar DNA quantitative variations may occur during meiosis in the plants in vivo.