Jan Salaj

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen‐activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress‐activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F‐actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co‐localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root‐derived cell culture. Loss of tip‐focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain‐of‐function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.

Initiation of embryogenic tissues and plantlet regeneration from somatic embryos of Pinus nigra Arn.

Embryogenic cultures were derived from immature zygotic embryos of Pinus nigra Arn. The zygotic embryos at the time of collection were at the precotyledonary stage of development. To stimulate the maturation of early somatic embryos, abscisic acid (ABA; 25 mg/l) combined with maltose (3, 6, 9%) were used. The maturation process appeared to be cell line dependent. The best results were obtained in cell line E15. In this cell line high concentrations (6 and 9%) of maltose improved somatic embryo maturation and well-formed cotyledonary somatic embryos developed. Two other cell lines tested (E7 and E16) showed prevailingly precotyledonary somatic embryo development. Although in cell line E16 in the presence of 9% maltose few cotyledonary embryos appeared, no plantlet regeneration occurred. Cotyledonary somatic embryos of cell line E15 germinated and regenerated plantlets with shoot and root meristem. The regenerated plantlets with root have been transferred to soil and at present are under observation.

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba x Abies cephalonica, and Abies alba x Abies numidica)

SummaryEmbryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.

DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture

Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

Developmental anatomy and ultrastructure of early somatic embryos in European black pine (Pinus nigra Arn.)

SummaryEmbryogenic callus cultures of European black pine (Pinus nigra Arn.) were established on megagametophytes containing zygotic embryos in early developmental stage. In addition to many elongated cells and disorganized growing clumps they contained early somatic embryos at various stages of development. At all stages of embryogenesis the embryos were organized as bipolar structures. Cell pairs composed of one isodiametric cell with dense cytoplasm and a second large vacuolated cell were the simplest bipolar system. The vacuolated cell underwent senescence. The cytoplasm-rich cell and its derivates divided transversally, resulting in several cytoplasmic cells arranged in row. An early embryonal cylindrical mass was formed by longitudinal division of the cells in a filament. Proximally localized cells in the early embryonal mass became vacuolized and elongated gradually giving rise to the secondary suspensor. Distal cells remained cytoplasmic in character and formed an embryonal mass along the axis of long early somatic embryos. Differences in the proportion of organelles and heterochromatin clumps, thickness of cell walls and number of plasmodesmata between cells at various stages of early somatic embryogenesis were described.

Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Summary Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin ( Abies alba x A. cephalonica ) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons ( Abies alba x A. cephalonica, Abies alba x A. numidica ), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures. The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons. Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.).

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.

Somatic Embryogenesis in Pinus Nigra Arn.

The European black pine (Pinus nigra Arn.) is one of the Slovakian pines involved in somatic embryogenesis study and belongs, taxonomically, to he section Pinus of the subgenus Diploxylon (Little & Critchfield, 1969). However, in spite of a common occurrence within the subsection Sylvestris, the species was shown to be sexually incompatible with the two additional species native to Slovakia, e.g. with Pinus sylvestris and Pinus mugo (Vidakovic, 1974; Kormutak & Lanakova, 1988). As a member of the subgenus Diploxylon, P. nigra shares the features postulated by Mirov (1967) for the genetics of a whole subgenus, e.g. a high irregularity in respect to hybridization. According to Vidakovic (1974), of the 13 hybridization attempts performed so far, only 6 interspecific crosses were successful, involving the combinations with taxonomically and geographically distant species such as Pinus resinosa, Pinus heldreichii, Pinus densiflora, Pinus thunbergii, Pinus tabulaeformis, and Pinus halepensis, respectively.

Dehydrin genes and their expression in recalcitrant oak (Quercus robur) embryos

In this work, three dehydrin genes, QrDhn1, QrDhn2, QrDhn3, were isolated from recalcitrant oak (Quercus robur). Their expression pattern was analyzed in both zygotic and somatic embryos as well as in vegetative tissues exposed to different kinds of abiotic stresses including desiccation, osmotic stress, and chilling. The QrDhn1 gene encoding for YnSKn type dehydrin was expressed during later stages of zygotic embryo development but in somatic embryos only when exposed to osmotic or desiccation stress. In contrast, the other two oak dehydrin genes encoding for putative Kn type dehydrins were expressed only in somatic embryos (both not-treated and osmotically stressed) and leaves of oak seedlings exposed to desiccation. Behavior of these genes suggests that different dehydrins are involved in processes of seed maturation and response to altered osmotic (water status) conditions in somatic embryos. Revealing further members of dehydrin gene family in recalcitrant oak might contribute to clarify non-orthodox seed behavior as well as identify mechanisms contributing to desiccation tolerance in plants.