L. Giorgetti

On the occurrence of somatic meiosis in embryogenic carrot cell cultures

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.

Ploidy reduction and genome segregation in cultured carrot cell lines. II. Somatic meiosis

Carrot cell lines W1 and W2 express permanently in culture a meiotic-like phenotpe, with apparent pairing and chiasmata formation comparable to meiosis during carrot microsporogenesis. The variant lines also show several variants of division in relation to the presence or absence of cytokinesis, nuclear fusion or spindle disturbance.The meiotic-like divisions can also be found in the abnormal structures, which are regenerated from these spontaneous variant lines. A possible role of the chromosome reducing mechanisms on carrot embryogenesis capacity and somaclonal variability is postulated.

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.

The Cell’s Commitment to Somatic Embryogenesis

Embryogenesis may be defined as the developmental program which, starting from two independent fertilization events, proceeds through coordinated stages to form a dormant embryo which is preserved and sheltered in the maternal ovary tissues by means of specific, and often elaborated structures, i.e., the seed and the fruit. In the life cycle of the flowering plant, this process plays a determinate role, gathering all the expectations for the future harvest and the performances of the forthcoming sporophytic organs. The functions of the maternal involucre and of agents or factors which may eventually influence the embryogenetic program, are largely unknown. Very little experimental work has been accomplished in this field, due to the small size of the zygote and its location deep within the maternal tissue. Notwithstanding these difficulties, the plentiful and accurate reports tracing the various stages of the embryological events, described with painstaking precision for most flowering plants, have to be acknowledged (Maheshwari 1950; Johri 1984).

Methylated DNA sequence extrusion during plant early meiotic prophase

Abstract Reproduction in plants has been thoroughly studied and abundant information is currently available in scientific literature. Here we present new data related to a process of DNA modulation along the meiotic steps, first described 50 years ago, but never further pursued. A time course of DNA content/nucleus by cytophotometric analysis, along the reproductive steps from Pollen Mother Cells (PMCs) to seedlings in barley and oil palm displays a significant modulation of nuclear DNA content that reaches its minimum at the zygote stage. Recovery of the DNA content/nucleus during seed germination stages has been demonstrated. Moreover, cytological and immunocytochemical studies indicate that methylated heterochromatic bodies are extruded from PMC nuclei in the progression of the pre-leptotene stage up to the bouquet. The heterochromatic and methylated status of the extruded bodies gives these data a perspective aligned with exciting current data relating to DNA methylation dynamics and chromatin modifications. Our hypothesis is that somatic cells committed to undergoing meiosis need to release the burden of accumulated somatic multicopy DNA sequences and regain totipotency before entering the process of gametogenesis. The event is interpreted as a mechanism for assuring a single copy DNA pairing and therefore avoiding the risk of unequal crossing-over. Methylation represents the evidence of RNA interference machinery at work, leading to heterochromatinization and extrusion of sequences. The recovery of the extruded DNA sequences along embryo development suggests the occurrence of gene amplification mechanisms.

Floral genes expressed in tomato hypocotyl explants in liquid culture.

SummaryThis paper confirms, at molecular level, previous data showing that small explants of many plants do form a floral meristem and express specific floral genes after only few days in culture. After 15–20 days of culture, small tomato hypocotyl explants develop differentiated structures often resembling primitive ancestral reproductive organs. Other specific reproductive functions such as chromosomal segregation (somatic meiosis) were also present and demonstrated by means of a cytological and histological analysis. By reverse transcriptase-PCR and in situ hybridization it was found that these structures are indeed able to express flower-specific genes. TheTM8 gene, a tomato gene that is expressed very early during floral development, is detectable on the proliferating hypocotyl explants during the first week of culture. TheMON9612 gene, which in vivo is expressed only by tomato pistils and ovules, is detectable on the ovulelike structures developed after 20 days of culture. The construction of transgenic tomato plants expressing theGUS gene under the control of the MON9612 promoter allowed us to follow the induction and the expression of this gene during explant proliferation and development of the flowerlike structures. These data confirm the hypothesis that a floral reprogramming can be induced in plant explants as a consequence of wounding and growth factors action. It appears to be an effort to survive stress by means of an unscheduled reproductive program.

Oil palm in vitro regeneration: microdensitometric analysis during reproduction and development

Abstract An extensive microdensitometric analysis was performed in oil palm Elaeis guineensis Jacq. during the establishment of a new embryogenic culture, on new regenerated plants at different times, and finally on seedlings obtained from adult regenerated normal and abnormal plants. Moreover nuclear DNA content was determined as well during the progression of meiosis. A variable loss of DNA content/nucleus in regenerated plants was demonstrated, in some cases very severe, depending mainly on the in vitro stationing of explants before the regeneration induction; DNA variation, but to less extent, was recorded also in seedlings from adult mother plants of the same genotype, in vivo and in vitro propagated. Considering normal and abnormal regenerated plants, DNA content variations did not seem to be necessarily connected to the abnormal phenotype. In addition stable and different variants in genome size could be obtained from in vitro culture of the same explant. Changes in the values of nuclear DNA content were determined during the meiotic prophase, confirming DNA extrusion processes also observed in other plant systems in vivo. Indeed the regeneration process may induce a quantitative DNA modulation/loss per cell in the regenerated plants but progressively DNA sequences should be regained before completion of the meiotic process. This information points to mechanisms of somaclonal variation.

Effect of white wheat bread and white wheat bread added with bioactive compounds on hypercholesterolemic and steatotic mice fed a high-fat diet

BACKGROUND The effects of white wheat bread and white wheat bread added with a bioactive compound mixture (Cyclanthera pedata, Glycine max, Monascus-fermented red mold rice, Cynara scolymus and Medicago sativa) were examined on hypercholesterolemic and steatotic mice, divided into four groups: control diet (CTR), high-fat diet (HFD), high-fat diet with white wheat bread added with 1.5 g kg(-1) of mixture (HFD+AB) and high-fat diet with white wheat bread (HFD+B). RESULTS Total serum cholesterol in the HFD+AB and HFD+B groups and hepatic triglycerides in the HFD+AB group decreased compared with the HFD group. Liver histology confirmed lower lipid drop accumulation in the HFD+AB group than in the HFD and HFD+B groups. HFD+AB caused a 7.0-fold increase and a 3.5-fold reduction in CYP7A1 and SREBP-1c gene expression respectively compared with the HFD group. Moreover, the HFD+B group showed a 2.2-, 8.4- and 1.5-fold increase in HMG CoA reductase, CYP7A1 and LDLr gene expression respectively compared with the HFD group. CONCLUSION Both the white wheat bread and the added white wheat bread induced cholesterol reduction by increasing CYP7A1. Moreover, the added white wheat bread improved steatosis by decreasing SREBP-1c gene expression.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia. Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen’s reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA+/DAPI- NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Analogies of Chromosome Reducing Events in Somatic Cultured Cells and in Microsporogenesis

Carrot somatic cells are considered totipotent,i.e committed to a determinate fate of development, since they can differentiate somatic embryos similar to zygotic embryos when the growth factors are withdrawn from the culture medium.