Kiem Tran Thanh Van

Morphogenesis in tobacco subepidermal cells: Putrescine as marker of root differentiation

We have used the tobacco thin cell layer ‘in vitro’ system to evaluate changes in polyamine titers as correlated with root differentiation and with variations in external pH during culture. We show that root differentiation in this system depends on both a rise in putrescine titers and a drop of pH, each of these two factors acting independently. With respect to polyamine titers, the most dramatic changes occur in the levels of putrescine liberated from perchloric acid-soluble conjugates. These titers increase from day 0 to day 7 of culture, reaching almost 2000 nmol g-1 fresh weight. Inhibition of putrescine biosynthesis prevents root initiation, while exogenous putrescine supply reverses this effect. We conclude that putrescine is a good marker for root differentiation.

Obtention of in vitro flowers from thin epidermal cell layers of Petunia hybrida (Hort.)

Abstract In vitro shoots and flowers are formed from thin epidermal cell layers of Petunia . Vegetative shoots are obtained in almost all genotypes but their number depends on the genotype. Floral shoots are obtained on the hybrids SK176 × Rly1, D + r67 × sfd1 and sfd1 × D + r67. In these hybrids, in vitro flowers are only formed when explants are excised from floral branches at a specific stage of their development. The precocity of in vitro shoots to flower depends on the origin of the explant. Floral buds are obtained in 6 weeks from explants excised at the 2nd internode whilst excised from the 3rd produce buds after 12 weeks. In vitro shoots formed on explants excised from 4th and 5th internodes can only be induced to flower if thin cell layers (tcl1) of these shoots are cultured. Micro-cuttings of the shoots (vs1) of tcl1 explants flower precociously. In vitro flowers of Petunia resemble flowers formed on plants cultivated in green-house. When anthers of these flowers are cultured (androgenesis in vitro), direct embryos have been obtained. These embryos develop into viable plants but their ploidy was not identified.

An Experimental Model for the Analysis of Plant/Cell Differentiation: Thin Cell Layer Concept, Strategy, Methods, Records and Potential

Plant differentiation including morphogenesis, growth, development and plant senescense result from complex interaction between different organs, tissues and cells. The analysis at the molecular level of the mechanisms of differentiation is difficult due to the lack of knowledge on i) the nature of the morphogenetic signal, ii) the perception and transduction mechanisms and iii) the localization of target cells and of responsive cells.

Study of Gene Expression During in Vitro Culture of Tobacco thin Cell Layers by two-Dimensional Electrophoresis of Proteins

Compared to animal systems, plant systems have tremendous potential for organ regeneration, embryogenesis from somatic cells, pollen grains, and ovules, for early sexual reproduction in in vivo conditions and in vitro conditions. Advanced progress already made in genetic manipulation technology would have led to important plant improvement if its feasibility was not limited to a small number of species. This limit is due to the difficulties in controlling in vitro plant regeneration via protoplasts, cells, tissues and/or organs culture of recalcitrant species such as monocotyledons, leguminous, and woody species which also have great economic importance. It is generally observed in recalcitrant species that regeneration of organs can be induced only during the juvenile phase. For example, mature tissues of woody plants lose their ability to differentiate organs in vitro. It is important to understand the phenomenon of maturation in recalcitrant species as it is important to understand the mechanisms of organ regeneration in recalcitrant, and nonrecalcitrant species. Progress is slow, because of the lack of regeneration /maturation mutants and because of the complexity of the eucaryotic genome. In species such as Arabidopsis for which the genome size is relatively reduced and for which several mutants are available, it still lacks developmental and regeneration mutants. There exist mutants with altered flower shape; however, these mutations are not suitable for studying the control of organogenesis.

In vitro flower formation from thin epidermal cell layers of a partial somatic hybrid between Petunia hybrida (Hort.) and Nicotiana plumbaginifolia (Viv.)

In vitro flowers have been obtained by culturing thin epidermal cell layers of a partial somatic intergeneric hybrid. The phenotype of these flowers differs from that of flowers formed on seed-grown plants (in situ flowers) and from that of flowers of either parental line. In addition, modifications in the phenotype were observed when cultures were sustained for more than four months. Dimorphic leaves present in juvenile and adult stages of mother plants of Nicotiana plumbaginifolia and the somatic hybrid were formed on different ends of the thin epidermal cell layers. No anomalies were observed during microsporogenesis and in the meiotic and mitotic figures of the somatic hybrid, which resembled those of Nicotiana plumbaginifolia.