Snežana Budimir

Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.).

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.

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.

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl-1 benzylaminopurine and 0.17 mgl-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.

Benzyladenine induction of buds and somatic embryogenesis in Picea omorika (Pancic) Purk.

Somatic embryogenesis and adventitious bud formation, initiated from shoot explants of Picea omorika is described. Benzyladenine (BA) as the only growth regulator, added to modified Von Arnold and Eriksson medium, induced formation of both adventitious buds and embryogenic tissue. Optimal BA concentration for bud induction was 4.5 μM and further bud development and plantlet formation was achieved on growth regulator-free medium. The embryogenic tissue formation was induced when the explants were first grown on the medium with high BA content (22.5 μM) and then transferred to medium without growth regulators. Subsequent proliferation of embryogenic tissue was accomplished by subculturing on medium containing 9 μM 2,4-dichlorophenoxyacetic acid and 4.5 μM BA, and further embryo development was achieved on medium with 12 μM abscisic acid. Embryos cultured on growth regulator-free medium formed roots and rooted plantlets were successfully established in soil in the greenhouse.

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.

Lenticel hypertrophy in shoot cultures of Ceratonia siliqua L.

Numerous white surface proliferations appeared in cultures of Ceratonia siliqua L. grown three to four weeks on medium containing 0.5 mg l-1 BA and 0.1 mg l-1 IBA. It was histologically confirmed that these proliferations were hypertrophied lenticels. Proliferations appeared first at the basal shoot internode and gradually spread acropetally, covering eventually the whole shoot except the uppermost internodes. Increase of BA concentration in the medium increased both the number of hypertrophied lenticels per shoot and the shoot multiplication index.