This 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. The TM8 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. The MON9612 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 the GUS 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.

6-Methoxymellein (6-MM) is the carrot phytoalexin and 6-hydroxymellein (6-HM) its immediate biosynthetic precursor. 6-MM is more toxic than 6-HM to the producing carrot cells, as demonstrated by the differential toxicity of these two dihydroisocoumarins to either colony forming units in solid medium or cell viability and mitotic activity in suspension cultures.

Totipotency is an unique property of plant cells. It is the capacity to repeat, starting from a single somatic cell, the developmental pathway normally followed by a fertilized egg in the ovary. This remarkable capacity, until now a puzzling phenomenon, is fully expressed when plant cells are cultured in vitro , allowing the regeneration of new plants completely modeled on their mother plant. The finding that transition from mitosis to meiosis may occur in cultured plant cells, and therefore possibly an alternative pathway of the mitotic process under stressing conditions, may offer a possible solution for the totipotency enigma. These data and the unexpected finding that plants regenerated from cultured plant cells have often deviated from the norm, have promoted the study of plant cell behavior in culture. This article gives details about the different deregulations of the cell cycle occurring in culture, in comparison with similar phenomena described in other organisms. The meiotic process, considered an alternative of the mitotic process when it occurs in somatic cultured plant cells, is discussed in detail, taking into account the importance, for evolutionary purposes, of this source of genetic variation. A new concept of cell totipotency is formulated, as expressed in plant cells when, by means of a meiotic-like process, they acquire a gametic condition.

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.

Cytological and histological analysis of hypocotyl expiants of many species revealed the occurrence of meiotic-like events (somatic meiosis and prophase reduction) located into structures differentiated around the vascular strands. These structures could be assimilated to primitive reproductive organs (pistil- and anther-like) containing pollen-like and embryo-sac like cells. Previous work demonstrated that segregating events in carrot are a prerequisite for the acquisition of totipotency Here the floral nature of the structures developed from hypocotyls in culture was demonstrated, at the molecular level, in expiants of tomato, Lycopersicon being a specie where floral specific genes are available. In situ hybridization experiments showed, in tomato hypocotyls cultured in vitro, the induction of two tomato floral specific genes: the TM8, a gene of the MAD family which in vivo is highly expressed specifically in floral meristems, and the MON 9612, a floral specific gene very tightly spatially and temporally regulated in tomato pistils. A study of the temporal pattern expression of these genes was performed by RT-PCR

Fusarium moniliforme endo-polygalacturonase and an elicitor preparation derived from the cell walls of Phythophthora megasperma induced in carrot cell suspension cultures both the accumulation of the carrot phytoalexin, 6-methoxymellein (6-MM), and the activity of 6-hydroxymellein O-methyltransferase (6-HM O-MT), an enzyme which specifically methylates the C-6-hydroxyl group in 6-hydroxymellein (6-HM) to give 6-MM. These fungal elicitors also stimulated a marked increase of phenylalanine ammonia-lyase (PAL) activity without affecting chalcone synthase (CHS) activity. A constitutive caffeic acid O-methyltransferase activity was identified in the same cultured carrot cells.

The temperature-sensitive carrot cell variant ts11c, arrested in somatic embryogenesis after the globular stage, was characterized. The sensitivity to a shift from 24° C (permissive temperature) to 32° C (non-permissive temperature) is greatest at the globular stage of embryogenesis, while cells proliferating in unorganized fashion and plantlets are not affected. Embryogenesis in ts11c is also arrested at the permissive temperature by replacement of conditioned culture medium with fresh medium. The timing of sensitivity of ts11c to medium replacement coincides with the sensitivity to temperature shift. Both sensitivities are recessive in somatic hybrids between ts11c and wild-type cells. Extracellular glycoproteins synthesized by ts11c at the non-permissive temperature contain much less fucose than those synthesized by the wild type. The glycoproteins synthesized by the variant under non-permissive conditions do not accumulate at the periphery of the embryo, as their wildtype counterparts do, but instead show a diffuse distribution throughout the embryo. The defect in ts11c can be fully complemented by the addition of extracellular wild-type proteins. A revertant of ts11c was isolated that simultaneously reacquired temperature insensitivity and normal glycosylation ability. Collectively, these observations indicate that ts11c is not able to perform proper glycosylation at the non-permissive temperature and suggest that the activity of certain extracellular proteins, essential for the transition of globular to heart stage somatic embryos, depends on the correct modification of their oligosaccharide side-chains.

6-Methoxymellein, a phytoalexin of carrot, and 6-hydroxymellein, its presumed biosynthetic precursor, were isolated from carrot root slices infected with Sclerotium rolfsii and Fusarium solani. Elicitation of both 6-methoxymellein and 6-hydroxymellein synthesis was achieved also in carrot cell suspension cultures by direct addition of pectinolytic enzymes such as Aspergillus niger pectinase highly purified Fusarium moniliforme endo-polygalacturonase (EC. 3.2.1.15). 6-Hydroxymellein was preferentially detected inside the cells while 6-methoxymellein was always found in the culture medium. Changes in pH values at the moment of elicitation of carrot cell suspension cultures influenced the amounts of both phytoalexins synthesized.

Polyamine content, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19) activities, as well as the effects of two inhibitors of putrescine synthesis, were studied during the preembryogenic phase and different stages of somatic embryogenesis in suspension cultures carrots (Daucus carota L.). In the preembryogenic phase α-difluoromethylornithine at 5mM, an irreversible inhibitor of ornithine decarboxylase, does not reduce growth but causes inhibition of putrescine accumulation; canavanine, a competitive inhibitor of arginine decarboxylase, completely blocks growth in the same phase. Difluoromethylornithine seems to prolong the commitment of the cells to embryogenesis during the preembryogenic phase, while canavanine inhibits the subsequent embryo differentiation. In general, a decrease in polyamine content and higher ornithine decarboxylase as compared to arginine decarboxylase activity is observed during the preembryogenic phase. Difluoromethylornithine does not affect ornithine decarboxylase in vivo, while both difluoromethylornithine and canavanine inhibit ornithine and arginine decarboxylase, respectively, in vitro. Spermidine and spermine contents, expressed on a per embryo basis, increase during the progression from globular to heart and torpedo stages of embryogenesis. While putrescine is the main polyamine in the heart stage, spermidine is more abundant in the torpedo stage. Embryos treated with difluoromethylornithine for 36h develop into normal plantlets at a higher frequency, and they are consistently larger than controls; the canavanine treatment blocks any further development of the embryos, which are always abnormal.

The phenomenon of habituation is considered in plant tissue cultures to be a real process of chemical tumorogenesis; the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca x N. langsdorffii nontumorous hybrid (NNT) during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.

The level of auxin - both natural and synthetic — in the medium has a strong effect on the level of 5-methyl-cytosine in the DNA of carrot cells in culture. This level may vary from approximately 15% to 70% of total cytosine without apparent effects on growth rate and cell morphology. No effect was seen with cytokinin. During somatic embryogenesis, in the absence of hormones, variations were seen in the level of methylation according to a characteristic pattern. If hypomethylation is induced with drugs such as azacytidine, ethionine or ethoxy-carbonyl-pyrimidine, embryogenesis is immediately blocked. A mutant was isolated which is resistant to the action of hypomethylating drugs. It shows variations in the methylation pattern and variations in indole-acetic acid metabolism. In addition its regeneration is often associated with the production of tumors.

Juvenile leaves of Cupressus arizonica Green (3–5 mm in length) from eight week old seedlings, were cultured on liquid medium supplemented with isopentenyladenine (2 mgl-1). Buds formed from the explants after three weeks of culture, but further growth occurred only after transfer to half-strength medium without plant growth regulators. Histological analysis at different times of culture, showed an early mitotic activity within transfusion tissue, followed by dedifferentiation of epidermal and mesophyll parenchyma cells at the basal zone of the leaves. The differentiation of vascular nodules always preceded bud formation. The difficulty of conifers to root and grow beyond plantlet stage is discussed.

We have studied the cytogenetic effect of 4-epoxyethyl-1,2-epoxy-cyclohexane (VCH-diepoxide) on Vicia faba and Allium cepa root tip meristems. Cytogenetic effect was estimated as percentage of micronuclei formed in interphase. We have shown that specific regions of M and S chromosomes of Vicia and allium telomeric regions as well were involved in the aberrations. Moreover we have demonstrated that DNA containing cytoplasmic structures defined ‘chromatin bodies’ and resembling chromocenters were extruded into the cytoplasm of both treated species. Thymidine post treatments, after the chemical exposure, prove a synergistic effect on the clastogenetic activities of VCH-diepoxide in V. faba and A. cepa.

The specificity of the clastogenetic effect of 4-epoxyethyl-1,2-epoxy-cyclohexane (VCH-diepoxide) on Vicia faba and Allium cepa has been investigated. Cytogenetic effect was estimated by Giemsa staining banding techniques on metaphase and anaphase analysis of chromatid and chromosome aberrations.