Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy.

In the present study a new luminescent dye 3-N-(2-pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3-aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.

Generation of interspecific hybrids of Trifolium using embryo rescue techniques.

The genus Trifolium Leguminosae (Fabaceae), commonly called clovers, includes 237-290 annual and perennial species, of which about 20 are important as cultivated and pasture crops. Taxonomic distribution supported by molecular analysis indicates that Mediterranean region is one of the main centers of distribution of the genus and also a center of domestication and breeding. Self-incompatibility is prevalent in the genus, controlled by a single, multiallelic gene expressed gametophytically in the pollen. It was suggested that hybridity did not play a major role in the evolution of the genus due to the poor crossability of the species under natural conditions. Interspecific hybridization in the genus Trifolium by conventional crossing techniques has been largely unsuccessful. Post-zygotic barriers appear to be a primary cause of the reproductive isolation, associated with endosperm disintegration and consequent abnormal differentiation and starvation of the hybrid embryo. As hybridization using conventional techniques has almost failed in Trifolium, embryo culture technique was used by breeders to obtain new combinations of interspecific hybrids. Embryo culture has been effectively used in developing interspecific hybrids in Trifolium ambiguum, T. pratense, T. montanum, T. occidentale, T. isthomocarpum, T. repens, T. nigrescens, T. uniflorum, T. sarosiense, T. alexandrinum, T. apertum, T. resupinatum, T. constantinopolitanum, T. rubens, and T. alpestre in various combinations. The successful embryo -rescue and development of hybrid plantlets requires skilled techniques of tissue culture and field practices. It includes hybridization in field; excision of hybrid embryos at appropriate stage; disinfection and culture in suitable culture media to allow maturation of embryo, multiplication of shoots, and rooting; hardening of the plantlets; inoculation with suitable Rhizobium culture; and transfer to field.

Ultrastructural changes of the egg apparatus associated with fertilisation of natural tetraploid Trifolium pratense L. (Fabaceae).

The aim of this study is to describe the ultrastructural changes of the egg apparatus associated with fertilisation of the natural tetraploid Trifolium pratense. The pollen tube enters one of the synergids through the filliform apparatus from the micropyle. Before the entry of the pollen tube into the embryo sac, one of the synergids begins to degenerate, as indicated by increased electron density and a loss of volume. This cell serves as the site of entry for the pollen tube. Following fertilization, the vacuolar organisation in the zygote changes; in addition to the large micropylar vacuole, there are several small vacuoles of varying size. Ribosomal concentration increases significantly after fertilisation. In T. pratense, ultrastructural changes between the egg cell and zygote stages are noticeable. Several marked changes occur in the egg cell because of fertilisation. The zygote cell contains ribosomes has many mitochondria, plastids, lipids, vacuoles. After fertilization, most of the food reserves are located in the integument in the form of starch. The zygote shows ultrastructural changes when compared to the egg cell and appears to be metabolically active.

Ultrastructural changes of the egg apparatus associated with fertilisation of natural tetraploid Trifolium pratense L. (Fabaceae)

The aim of this study is to describe the ultrastructural changes of the egg apparatus associated with fertilisation of the natural tetraploid Trifolium pratense. The pollen tube enters one of the synergids through the filliform apparatus from the micropyle. Before the entry of the pollen tube into the embryo sac, one of the synergids begins to degenerate, as indicated by increased electron density and a loss of volume. This cell serves as the site of entry for the pollen tube. Following fertilization, the vacuolar organisation in the zygote changes; in addition to the large micropylar vacuole, there are several small vacuoles of varying size. Ribosomal concentration increases significantly after fertilisation. In T. pratense, ultrastructural changes between the egg cell and zygote stages are noticeable. Several marked changes occur in the egg cell because of fertilisation. The zygote cell contains ribosomes has many mitochondria, plastids, lipids, vacuoles. After fertilization, most of the food reserves are located in the integument in the form of starch. The zygote shows ultrastructural changes when compared to the egg cell and appears to be metabolically active.