Somatic embryogenesis was initiated from immature embryos of Abies concolor (Gord. et Glend), A. numidica De Lann. and A. cilicica Carr., A. alba Mill. as well as in hybrid fir A. cilicica × A. nordmanniana. Soluble protein profiles and peroxidase activity were compared in developing zygotic and somatic embryos of silver fir (A. alba Mill.). On the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins a high degree of homology was established between the two types of embryos. A higher peroxidase activity was registered throughout zygotic embryogenesis than during somatic embryo development but the opposite was true at the stage of mature embryos. Isoperoxidase composition reflected more efficiently the developmental stages of zygotic embryogenesis than those of somatic embryogenesis.

The height growth dynamics of three interspecific crosses with Greek fir (Abies cephalonica Loud.) acting as mother and a control variant from self-pollination were followed throughout a period of 30 years. The hybrid combination A. cephalonica x A. nordmanniana exhibited enhanced growth relative to the selfed control at stage of 4-year old seedlings already, whereas the hybrid A. cephalonica x A. alba only at stage of 9-year old saplings. At age of 30, the heterotic growth persisted only in the latter, the hybrid A. cephalonica x A. nordmanniana was comparable in height growth parameters with a selfed control. The interspecific cross A. cephalonica x A. numidica was remarkable by its accelerated growth during early stages of the development. At age of 4 years it has surpassed in height growth both A. cephalonica x A. alba and control but has declined profoundly during subsequent stages of its development. A conspicuous feature of the hybrid needle anatomy were abundant resin canals and reduced size of some anatomical traits of its needles. In spite of variable growth potentials, all the three interspecific hybrids are recommended for planting in Slovakia, especially at densely polluted areas where domestic silver fir cannot withstand emission pressure.

Environmental scanning electron microscopy enables the investigation of uncoated pine early embryogenic tissue samples in situ. The samples were examined under low vacuum conditions (air pressure 550 Pa) at a temperature of around -18°C by the AQUASEM II noncommercial environmental scanning electron microscope. The native extracellular matrix surface network was imaged by the environmental scanning electron microscope and in dark field mode of the optical microscope too. The backscattered electron detector disclosed brightness loci in the cells of early embryogenic culture. This work shows images of native pine embryogenic tissues. The continuity of extracellular matrix with structural integrity of plant organism is discussed.

KormuťáK, A., ČAmeK, V., BrAnná, m., ČelKoVá, m., VooKoVá, B., mAňKA, P., GAlGóci, m., BoleČeK, P., Gömöry, D. 2013. Introgessive hybridization between Scots pine and mountain dwarf pine at two localities of northern Slovakia. Folia oecol., 40: 201–205. Introgressive hybridization within hybrid swarm populations of Pinus sylvestris and P. mugo was analyzed in the localities Medzi Bormi and Sokolie, northern Slovakia, using species-specific trnV-trnH/Hinf I restriction profiles of chloroplast DNA of needles and zygotic embryos. The presence of trees with both P. sylvestris and P. mugo haplotypes indicates hybrid nature of the swarms. Molecular analysis revealed a relatively high rate of introgressive hybridization between putatively hybrid swarm individuals and the parental trees of the pure species P. sylvestris and P. mugo growing in the same localities. The proportion of hybrid seeds with P. sylvestris pollen donor averaged at 30.3%, whereas those with pollen donor of P. mugo at 19.8%. The results are discussed within context of the established crossability relationship between P. sylvestris and P. mugo.

This paper provides results of somatic embryogenesis study in our laboratory. General description of somatic embryogenesis (SE) induction, maturation of somatic embryos and plantlets regeneration of the Abies species, followed by a comparisons of some characteristics of zygotic and somatic embryos, seedlings and emblings (somatic seedlings). Own results are supplemented with some literature data. Also aplication of SE for improving of plantlet regeneration of elite fir trees from Dobroc primeval is described as well as initiation of the SE from seeds of incompatible crossings of firs where zygotic embryos abort usually several weeks after pollination.

Differential inhibition in growth of fungus Phaeolus schweinitzii inoculum was tested with somatic embryos of two cell lines of silver fir (Abies alba Mill.) by the method of dual cultures with pre-cultivation of somatic embryos and subsequent removal. It was confirmed that inhibitory substances from pre-cultivated somatic embryos L27 spreading to agar cultivation medium by simple diffusion cause greater inhibition of tester mycelial growth during subsequent co-cultivation compared to somatic embryos L122. Characteristics of embryogenic cell lines L27 and L122, from which somatic embryos were formed, are provided. Increased levels of peroxidase activity characterized the cell line L27. Natural non-induced constitutive defense of plant somatic embryos established by quantitatively measured restriction of growth in tester Phaeolus schweinitzii was discussed.

Crossability relationships between six species of the Mediterranean, North American and Asian firs was tested using Abies alba and A. nordmanniana as female parents and A. alba, A. numidica, A. procera, A. grandis, and A. holophylla as pollen parents. An overwhelming majority of the crosses attempted was found to be compatible. In particular, it is true of the A. alba cross with A. numidica and those of A. nordmanniana with A. alba, A. numidica, A. procera, and A. holophylla. The crossing A. nordmanniana × A. grandis was the only exception producing empty seeds. Cytological study revealed the gametophytic incompatibility to be responsible for reproductive isolation of these species. At seedling level, all the interspecific crosses of A. nordmanniana surpassed in height growth self-pollinated control. The cross A. alba × A. numidica was comparable in this respect with control variants from open and self-pollination. Except for height growth, some characteristics of needle stomata are provided for individual crosses. The crosses A. nordmanniana with A. procera and A. holophylla represent unique interspecific combinations whose existence has not been reported yet. Based on needle stomata characteristics, the potential for increased resistance and drought tolerance of the hybrids with A. numidica involved as parental species is discussed.

Defence reactions of desiccated cotyledonary somatic embryos and mature zygotic embryos of Abies numidica were tested by dual cultures with tester, fungus Phaeolus schweinitzii. Both types of embryos expressed defence reactions. Mutual comparisons of zygotic and somatic embryos showed important differences between defence reactions against mycelial growth towards these embryos. Greater defence reactions were observed in zygotic embryos relative to defence found in somatic embryos.

This article describes the usage of non-commercial environmental scanning electron microscope (ESEM) for the visualization of plant extracellular matrix in Abies alba and Abies numidica. Non sputter-coated samples free of using the common fixation technique observed at the relatively low humidity of the air environment with the pressure 550 Pa and the low temperature of the sample from −18 to −22°C give surprisingly very good results that show the natural structure of the tissues. This seems to be generally applicable. Moreover, a specially designed ionization detector of secondary electrons and a YAG:Ce3+ detector of backscattered electrons were used for better comparison.

We used artificial hybridization to study the crossability of the noble fir (Abies procera) with Manchurian fir (A. holophylla) and Caucasian fir (A. nordmanniana), and found compatibility between A. procera of North American origin and the Asian species A. holophylla as evidenced by the 14% fraction of filled seeds obtained in A. procera x A. holophylla crossing. Crossing of A. procera with the Mediterranean species A. nordmanniana failed completely, producing only empty seeds.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Defense of desiccated cotyledonary somatic embryos and mature zygotic embryos of Abies numidica was tested in vitro by dual cultures with tester, fungus Phaeolus schweinitzii.Both types of embryos expressed defense reactions manifested by inhibited growth of fungal tester towards the embryos.Mycelial growth was described by logistic sigmoid growth model with a single asymptote.Mutual comparisons of mycelial growth in presence of zygotic and somatic embryos showed signifi cant diff erences in parameters of mycelium growth curves towards the embryos.Larger defense reactions were observed in zygotic embryos relative to somatic embryos and unlimited control cultivations without embryo.The possible role of auxin in the defense response of plant embryos is discussed.

Pollen viability in three hybrid swarm populations of Pinus mugo x P sylvestris and in control populations of P. mugo in northern Slovakia was studied throughout two successive years Germination percentage and pollen tube length were used as the principal parameters of pollen viability. In 2007, a slight reduction of pollen germination was recorded in hybrid swarms on the localities Habovka and Sucha Hora in comparison with the adjoining population of P. mugo from Rohace Statistically significant differences between individual populations were revealed in pollen tube length only In 2008, a pollen of improved quality was produced in all the populations tested. Pollen germination of hybrid swarms in Habovka, Sucha Hora and Tisovnica was comparable in the respective year with control population of P mugo from Vratna valley. At the pollen tube level the hybrid swarms in Habovka and Sucha Flora deviated considerably from control population by their shorter pollen tubes. Obtained results are taken as an evidence supporting partial reduction of pollen viability in the respective hybrid swarms. Annual variation in pollen viability is supposed to be conditioned climatically

The effect of maturation pretreatment on development and growth of Abies numidica De Lann. somatic embryos was studied. The most beneficial was pre-culturing on Schenk and Hildebrandt medium without growth regulator for 2 weeks. Dry mass accumulation of emblings was lower than that of seedlings after 50 d of culturing. Contents of microelements in seedlings were higher than in emblings, but macroelements contents were higher in emblings. Contents of chlorophyll a and chlorophyll b in cotyledons were higher in seedlings than in emblings while no qualitative differences were detected between the protein profiles of seedlings and emblings.

Possibility to improve plantlet regeneration from Abies alba Mill. open-pollinated families of 4 trees in Dobroč primeval and 3 trees in managed forest was studied. Immature zygotic embryos were cultured in order to obtain initiation of embryogenic tissue. Totally, three from the families of the managed forest (57%) and two from the primeval families (50%) responded to initiation condition. Initiation frequencies among families ranged in managed forest: 4.5–56.2%, primeval: 5.4–16.8%. Maturation ability was shown by 77.3% of the primeval cell lines, 36.4% cell lines produced cotyledonary somatic embryos. In managed forest, in 62.5% of the cell lines embryo maturation was observed. Cotyledonary embryos developed only in 15% of cell lines. Regenerants were obtained from 9 cell lines of primeval and from 6 cell lines of managed forest. Biochemically, the mature somatic embryos were characterized by the variation in soluble and protein profiles. The corresponding profiles of insoluble proteins exhibited uniform pattern. The variation was characteristic for somatic embryos of individual cell lines rather than for the primeval and managed stands. Enzymatically, no indications were obtained supporting higher metabolic potential of somatic embryos derived from zygotic embryos of silver fir primeval stand than in somatic embryos originating from the trees of managed stand.

Crop characteristics involving cone size, average number of seeds per cone, and seed germinability have been studied for three hybrid swarm populations of Pinus mugo and P. sylvestris, two pure P. mugo populations and one pure P. sylvestris population, in northern Slovakia. Generally, reproduction capability characteristics were significantly reduced in hybrid swarms in relation to the control populations. Although there were no consistent differences between hybrid swarms and control populations in the average number of seeds per cone or cone size, seed germination rates and full seed production in hybrid swarms were lower than for the control population of P. sylvestris and one of the pure populations of P. mugo. Based on these data a conclusion has been drawn postulating partial rather than full fertility of the hybrid swarm populations of P. mugo and P. sylvestris.

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba x A. cephalonica (cell lines AC2, AC78) and Abies alba x A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l(-1)). The T-DNA of plant transformation vector contained the beta-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l(-1). GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.

Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship between Pinus sylvestris and Pinus mugo.

The present study aimes at initiating somatic embryogenesis in immature seeds of incompatible crossings of firs where zygotic embryos abort usually several weeks after pollination. The induction of embryogenic tissue was rather rare and occurred with frequencies of 0.64-1.6% in A. nordmanniana x A. concolor 0.69-3.82% in A. nordmanniana x A. veitchii, 5.55% in A. concolor x A. veitchii, 0.64-1.60% in A. nordmanniana x A. concolor and 1.23% in A. pinsapo x A. veitchii. The initiation of embryogenic tissue from other hybrid fir seeds was not achieved. Maturation up to cotyledonary stage of development was achieved only in A. nordmanniana x A. veitchii somatic embryos. The hybrid nature of the somatic embryos was proved by restriction analysis of chloroplast DNA.

Peroxidase activity was monitored during somatic embryogenesis of white fir (Abies concolor Gord. et Glend) starting from a non-embryogenic callus. Results revealed profound differences between non-embryogenic and embryogenic calli with an elevated level of enzyme activity in non-embryogenic ones. Precotyledonary, early cotyledonary and late cotyledonary stages of somatic embryogenesis were characterized by a substantially reduced peroxidase activity compared to callus tissues and regenerated plantlets. Changes in peroxidase activity are as a rule paralleled by variation in isoenzyme composition. The utility of the enzyme in the induction stage of somatic embryogenesis in white fir is proposed.