Branka Uzelac

Origin and development of secondary somatic embryos in transformed embryogenic cultures of Medicago sativa

Non-transformed and transformed embryogenic cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83), long-term maintained on growth regulator-free medium, were histologically analyzed. In all examined cultures, somatic embryos at various stages of development were observed and secondary embryos were formed in the cotyledonary, hypocotylary and radicular region of the primary embryos. Detailed histological analysis of the torpedo shape somatic embryo revealed that secondary somatic embryos arose directly from single epidermal cells of hypocotylary axis after an unequal periclinal division. Bipolar proembryos were composed of one smaller cytoplasm rich cell and one larger more vacuolated cell. Further cell division pattern was similar for both non-transformed and transformed embryos. However, multicellular origin of secondary embryos in a direct process and even from callus can not be excluded.

In situ detection of programmed cell death in Nicotiana tabacum leaves during senescence

Leaf senescence is a highly regulated developmental process, during which cell constituents are dismantled in an ordered progression. The final death phase of senescence is a type of programmed cell death (PCD). We showed DNA fragmentation characteristic for PCD by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay not only in senescing leaves but also in young, still developing tobacco leaves.

Micropropagation of Pinus peuce

In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.

Characterization of natural leaf senescence in tobacco (Nicotiana tabacum) plants grown in vitro

Leaf senescence is a highly regulated final phase of leaf development preceding massive cell death. It results in the coordinated degradation of macromolecules and the subsequent nutrient relocation to other plant parts. Very little is still known about early stages of leaf senescence during normal leaf ontogeny that is not triggered by stress factors. This paper comprises an integrated study of natural leaf senescence in tobacco plants grown in vitro, using molecular, structural, and physiological information. We determined the time sequence of ultrastructural changes in mesophyll cells during leaf senescence, showing that the degradation of chloroplast ultrastructure fully correlated with changes in chlorophyll content. The earliest degenerative changes in chloroplast ultrastructure coinciding with early chromatin condensation were observed already in mature green leaves. A continuum of degradative changes in chloroplast ultrastructure, chromatin condensation and aggregation, along with progressive decrease in cytoplasm organization and electron density were observed in the course of mesophyll cells ageing. Although the total amounts of endogenous cytokinins gradually increased during leaf ontogenesis, the proportion of bioactive cytokinin forms, as well as their phosphate precursors, in total cytokinin content rapidly declined with ageing. Endogenous indole-3-acetic acid (IAA) levels were strongly reduced in senescent leaves, and a decreasing tendency was also observed for abscisic acid (ABA) levels. Senescence-associated tobacco cysteine proteases (CP, E.C. 3.4.22) CP1 and CP23 genes were induced in the initial phase of senescence. Genes encoding glutamate dehydrogenase (GDH, E.C. 1.4.1.2) and one isoform of cytosolic glutamine synthetase (GS1, E.C. 6.3.1.2) were induced in the late stage of senescence, while chloroplastic GS (GS2) gene showed a continuous decrease with leaf ageing.

Secondary somatic embryogenesis versus caulogenesis from somatic embryos of Aesculus carnea Hayne.: developmental stage impact

Somatic embryos of red horse chestnut, derived from cultures maintained through repetitive somatic embryogenesis for a few years, were subjected to induction of secondary regeneration. The embryos were divided in four classes on the basis of their size (I-1, II-5, III-10 and IV-30 mm), and sub-cultured on MS media containing 0, 1, 5 or 10 μM kinetin (Kin) or benzyladenine (BA). The pathway of secondary regeneration, somatic embryogenesis or caulogenesis, depended on the primary somatic embryo (PSE) stage of development. The embryogenic capacity declined and bud-forming capacity increased with the degree of PSE maturity. The PSE of the Classes I and II produced only secondary somatic embryos (SSE), the Class III PSE formed both SSE and adventitious buds, whereas the Class IV PSE developed almost solely adventitious buds. The process of secondary somatic embryogenesis was most effective in the Class II PSE at 5 μM BA, and the process of adventive organogenesis was most effective in the Class IV PSE at 10 μM BA. On plant growth regulator (PGR)-free medium, PSE of A. carnea followed the same pattern of adventive regeneration, as those cultured on cytokinin containing media. The cytokinins only amplified the response, in a certain range of concentrations. BA promoted bud induction at a much higher rate than Kin, while their embryogenic effect was similar.

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary.In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development.

Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

Abstract Study describes protocols for in vitro propagation of Gentiana utriculosa L. via axillary shoot multiplication and indirect somatic embryogenesis. Shoot cultures were established from seedling epicotyl explants cultured on MS medium supplemented with 0.25 mg L−1 BA and 0.1 mg L−1 IAA. Medium containing 2% sucrose and 0.2 mg L−1 BA improved multiple shoot production, providing 2.3 shoots per explant. The highest rooting (29.6%) was obtained on medium with 1/2 MS mineral salts and 0.5 mg L−1 NAA. Somatic embryogenesis was induced using different explants, including immature seeds as well as leaves and roots from shoot cultures. Following auxin treatment with either 1.0 mg L−1 2,4-D (immature seeds and leaves) or 0.1 mg L−1 NAA (roots), explants produced embryogenic calli which upon transfer to plant growth regulator-free medium allowed embryo conversion into plantlets. The best embryogenic response (82%) was obtained in calli derived from leaves cultured with their abaxial surface in contact with medium, whereas the highest embryo conversion rate (68%) was recorded for calli induced on immature seed explants. Histological analysis in all explant types revealed development of proembryogenic cell complexes at callus periphery, giving rise to somatic embryos. The presence of embryos at various stages of development indicated asynchronous somatic embryogenesis in G. utriculosa. Derooted embryo-derived plantlets placed on medium with 0.2 mg L−1 BA multiplied further as shoot cultures.

Bosnian Pine Pinus heldreichii Christ.

Bosnian pine (Pinus heldreichii Christ.) is a Tertiary relict subendemic to the mountains of Balkan Peninsula and a small area in southern Italy.

Morphogenesis and developmental ultrastructure of Nicotiana tabacum short glandular trichomes

The anatomy and ultrastructure of the short glandular trichomes occurring on young expanding leaves of Nicotiana tabacum were investigated using light and transmission electron microscopy. The objective of the present research was to characterize the cellular changes that occur during morphogenesis of short glandular trichomes, from initiation to senescence. Ultrastructural analysis of their secretory cells revealed characteristics common to gland cells: numerous mitochondria in highly organized cytoplasm, large nuclei, and an elaborate network of endoplasmic reticulum. Initial changes in nuclear and plastidial organization were observed at a more advanced secretory stage, marking the onset of senescence. During trichome senescence, gradual reduction of the cytoplasm density occurred along with structural changes of the plastids and the tonoplast. As a result of inward blebbing of the cytoplasm into the vacuole, membrane bound vesicular structures appeared in the vacuolar space. At the late secretory stage, marked by an increase in vacuolation and extraplasmic space, degenerative changes included further fragmentation of the cytoplasm and deterioration of the tonoplast. Multimembrane myelin bodies observed in the vacuolar space were indicative of membrane digestion although plasma membrane did not appear massively degraded.

Effect of plant growth regulators on leaf anatomyof the has mutant of Arabidopsis thaliana

In this study, the effect of plant growth regulators on leaf morphogenesis of the recessive T‐DNA insertion mutant of Arabidopsis thaliana was analyzed. The morpho‐anatomical analysis revealed that leaves of the has mutant are small and narrow, with lobed blades and disrupted tissue organization. When has plants were grown on the medium supplied with plant growth regulators: benzylaminopurine (BAP) or ethylene precursor, 1‐aminocyclopropane‐1‐carboxylic acid (ACC), the leaf anatomy was partially restored to the wild type, although plants still exhibited morphological abnormalities.