Zoltán Kristóf

Improvement of isolated microspore culture of pepper ( Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.

Mechanoreceptor Cells on the Tertiary Pulvini of Mimosa pudica L.

Special red cells were found on the adaxial surface of tertiary pulvini of Mimosa pudica and experiments performed to determine the origin and function of these cells. Using anatomical (light, scanning electron and transmission electron microscopy) and electrophysiological techniques, we have demonstrated that these red cells are real mechanoreceptor cells. They can generate receptor potential following mechanical stimuli and they are in connection with excitable motor cells (through plasmodesmata). We also provide evidence that these red cells are derived from stomatal subsidiary cells and not guard cells. As histochemical studies show red cells contain tannin, which is important in development of action potentials and movements of plants. These cells could be one of unidentified mechanoreceptors of mimosa.

Isolation and osmotic relations of developing megagametophytes of Torenia fournieri

Abstract A new method is reported to isolate and handle living megagametophytes of Torenia fournieri at any developmental stage. The stages were determined using light microscopy and delimited by correlating floral morphological traits. When significant changes in the osmotic pressure were found during development, enzyme solutions contained different concentrations of osmoticum. Osmotic pressure is lowest in the megaspore, increases until the four-nucleate stage and then gradually decreases until complete embryo sac formation. In enzymatic solutions containing appropriate concentrations of osmoticum, protoplasts of megaspores, two-, four-, eight-nucleate embryo sacs, egg cells, synergids and central cells were successfully isolated. The living protoplasts were collected by micromanipulator, transferred into microdroplets and tested for viability.

Morphological characterisation of wheat (T. aestivum L.) egg cell protoplasts isolated from immature and overaged caryopses

Abstract The morphological features and fine structure of wheat egg cell protoplasts isolated from premature (3 days prior to anthesis) and overaged (12 days after anthesis) caryopses were compared. Except for shape, the young egg cell protoplast showed the same morphological characteristics as the egg cell in planta at the time of anthesis. Young and adult egg cell protoplasts were spherical in shape. Polarity could not be identified exactly with the methods used. During aging, the egg cell increased considerably in volume. The adult egg showed the typical features (membrane blebbing, autophagous vacuoles) of programmed cell death. It appears that after a long life-span (about18 days) cells of the female gametophyte undergo apoptosis.

A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces

Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Seri-DL-Alam)] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss® bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.

Comparative studies on the male and female gametophyte development in three different Triticum species

Abstract The in planta and in vitro development of the male and female gametophytes of three Triticum species ( T. aestivum cv. Mv 15/6 n /, T. araraticum /4 n / and T. monococcum /2 n /) was traced from just after meiosis until the development of the mature pollen or embryo sac. The dynamics of differentiation in the male gametophytes was not influenced by the different ploidy levels up to the tricellular stage. The maturing process of differentiated haploid male gametophytes, resulting in the shedding of mature pollen from the anthers, proceeds most rapidly in the hexaploid species. The development of the initial microspore slowed in vitro, remaining in the uninucleate stage for a longer period, with a delay in first pollen mitosis compared to the control. There was no substantial difference between the three species in the dynamics of in vitro pollen development. In all cases the uninucleate microspore developed into a functional tricellular pollen grain within a week. However, there were differences in the lengths of the various developmental stages under in vitro and natural conditions. The seven-cellular female gametophyte developed far more rapidly than the male gametophyte, which means that the mitotic divisions leading to the differentiation of male and female gametophytes were not synchronised within the plants. In species with lower ploidy levels macrosporogenesis took longer than microsporogenesis, and the development of the seven-celled embryo sac was completed 2 days later than in hexaploid species. The differing ploidy levels also influenced the size of the embryo sac. The embryo sacs achieved their maximum dimensions 2–3 days prior to male gametophyte maturation. These observations raise several questions as to when gametophytes can be regarded as functionally mature, and which phase of gametophytic development is most suitable for cell manipulation.

Development and isolation of the male gametophyte of Torenia fournieri

New methods were developed to isolate the male gametophyte of Torenia fournieri at any developmental stage. The stages were defined by light microscopic studies and identified by correlating morphological traits of the flower buds. Enzyme solutions were used to isolate gametophytic cells. Preliminary studies were carried out to determine the components of the cell wall, and the optimal osmotic pressures of the appropriate enzyme solutions were adjusted to the different developmental stages. We managed to isolate diploid microsporocytes, haploid microspores, cells of young and mature pollen grains, and sperm cells from growing pollen tubes. Isolated protoplasts were collected in microcapillaries to prepare them for further studies.