Andrej Kormutak

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.

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.

SOMATIC EMBRYOGENESIS IN HYBRID FIRS

The genus Abies is represented by more than 40 species widespread in North and Central America, Eurasia and North Africa. In Europe, out of 10 Abies species only Abies alba (silver fir) is widely distributed in the Central European mountains, Balkan, Apennines chain and in the Pyrenees while the others are distributed in small areas around the Mediterranean region.

Secondary Somatic Embryogenesis in Abies numidica

The induction of secondary somatic embryogenesis in Abies numidica De Lann. was achieved. Precotyledonary, cotyledonary, and desiccated cotyledonary embryos were used as explants. Cotyledonary embryos before desiccation were the most suitable. The most beneficial was induction medium Schenk and Hildebrandt (SH) with 1 mg dm−3 thidiazuron and 1000 mg dm−3 myo-inositol. Initiation frequency was from 1 to 34 %. Maturation of somatic embryos was achieved on modified Murashige and Skoog medium supplemented with 40 g dm−3 maltose, 100 g dm−3 polyethylene glycol-4000 and 10 mg dm−3 abscisic acid. Mature somatic embryos after three weeks of desiccation germinated on SH medium with 10 g dm−3 charcoal and 10 g dm−3 sucrose.

Peroxidase activity in non-embryogenic and embryogenic calli and in developing somatic embryos of white fir (Abies concolor Gord. et Glend).

ABSTRACT 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.

Ultrastructural and molecular study of plastid inheritance in Abies alba and some Abies hybrids

Abstract The ultrastructure of egg cells in Abies alba was examined to elucidate the lack of maternal inheritance of plastids. Before fertilization, maternal plastids are absent in the perinuclar zone containing mainly mitochondria and smooth endoplasmic reticulum. During egg cell development the maternal plastids are transformed into large inclusions which are situated mostly towards the periphery of the egg cell, and finally disintegrate. As a consequence, they do not participate in zygote formation.RFLP analysis of cpDNA of parental trees and their F1 interspecific hybrids (A. alba×A. numidica, A. alba×A. nordmanniana, A. nordmanniana×A. Alba) using HindIII and BamHI showed a paternal mode of cpDNA inheritance. Paternal inheritance has also been found with PCR/RFLP analysis of cpDNA from parental trees and their hybrids (A. alba×A. pinsapo, A. pinsapo×A. alba, A. pinsapo×A. numidica) using ApaI and HaeIII digests, as well as in the crosses of A. cephalonica×A. nordmanniana, A. nordmanniana×A. cephalonica, A. cephalonica×A. numidica using TagI digests.

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between Pinus sylvestris and Pinus mugo and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.

Seed quality in hybrid swarm populations of Pinus mugo Turra and P. sylvestris L.

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.

Abortive embryogenesis in hybrid swarm populations of Pinus sylvestris L. and Pinus muga Turra

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.

A comparison of normal callus culture and habituated ESM of silver fir (Abies alba mill.) for defense reactions to the fungus Phaeolus schweinitzii

Normal callus culture and habituated embryogenic suspensor mass (ESM) of silver fir (Abies alba Mill.) were compared enzymatically using three enzyme systems. Significant reduction of peroxidase activity, more intensive glutamate dehydrogenase activity and higher number of isoenzymes of non‐specific esterase were found in habituated ESM as compared with the normal calli. Defense reactions of normal callus culture and habituated ESM were tested by a simple method of dual cultures. Habituated ESM showed strengthened defense reactions against tester Phaeolus schweinitzii. Reasons of this phenomenon are discussed.