Paweł M. Pukacki

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichters (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichters medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.

The influence of ultraviolet-B radiation on the growth, pigment production and chlorophyll fluorescence of Norway spruce seedlings

Norway spruce (Picea abies (L.)Karst.) from seven seed sources was grown in a greenhouse with 8.3 and 14.7 kJ·m−2·d−1 m UV-BBE (biologically effective UV-B: 280–320 nm) irradiation, and with no supplemental irradiation as control. The seedlings total biomass (dry weight) and shoot growth decreased with high UV-B treatment but spruce from low elevation seed sources were more affected. The seedlings grown at the highest UV-B irradiance (14.7 kJ·m−2·d−1) showed from 5 to 38% inhibition of total biomass and 15 to 70 % shoot growth inhibition. Norway spruce populations from higher altitude seed sources manifested greater tolerance to UV-B radiation compared to plants from low altitudes. Changes in phospholipids and protective pigments were also determined. The plants grown at the lower UV-B irradiance (8.3 kJ·m−2·d−1) showed greater ability to concentrations UV-B-absorbing pigments then control plants. Chlorophyll a fluorescence parameter Rfd, (Rfd=(Fm-Fs)/Fs) showed a significant decrease in needles of UV-B treated plants and this correlated with the altitude of seed source. Exposure to UV-B affect levels of the ratio of variable to maximum fluorescence (Fv/Fm). Results from this study suggest that the response to increased levels of UV-B radiation is depended upon the ecotypic differentiation of Norway spruce and involved changes in metabolites in plant tissues.

Effect of water deficit on oxidative stress and degradation of cell membranes in needles of Norway spruce (Picea abies)

AbstractWe studied the impact of mild and severe drought stresses for 42 days and rehydration for 21 days on 4-year-old seedlings of Norway spruce. Water relations in spruce tissues were determined on the basis relative water content of needles and shoot water potential (Ψshoot). During the stress, we measured the level of: reactive oxygen species (ROS), antioxidants, and degradation of cell membranes. In the seedlings subjected to severe stress, Ψshoot decreased to −2.4 MPa, while in those subjected to mild stress, to −0.8 MPa. After rehydration, shoot water potential increased, but did not reach the control level. Water deficit caused oxidative stress, reflected in an increased production of ROS: superoxide anion radical ( % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaeWaaeaada% qfqaqabSqaaGqaaiaa-jdadaahaaadbeqaaiabgkci3oaaCaaabeWc% baGaaeylaaaaaaaabeqdbaGaa83taaaaaOGaayjkaiaawMcaaaaa!3BE2!

Deep supercooling of shoot and bud tissues of Picea abies

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Long-term implications of industrial pollution stress on lipids composition in Scots pine (Pinus sylvestris L.) roots

) and hydrogen peroxide (H2O2). Their concentrations in needles were the highest in seedlings subjected to severe stress, where they exceeded the control level by 116% and 30%, respectively. During rehydration, the differences in ROS levels between treated and control seedlings diminished. Oxidative stress causing degradation of cell membranes included: de-esterification of phospholipids, oxidation of fatty acids, and increase in concentration of malondialdehyde, as their permeability to ions increased by 125%. In the defence against the oxidative stress in needles, an important role was played by low-molecule antioxidants such as glutathione, ascorbic acid, flavonoids, α-tocopherol and antioxidant enzymes. An increase in intensity of water deficit caused a significant reductio in the level of low-molecular antioxidants, which attests to their utilization during the process of scavenging for free radicals. Water deficit at Ψshoot=−1.7 MPa caused a decline in ascorbic acid level by 37% in needle cells. An effective defensive mechanism removing the excess of ROS was also reflected in the activity of the main enzymes of oxidative stress: superoxide dismutase (SOD) and guaiacol peroxidase (PO). As a result of water deficit, SOD activity increased by 80 %, while PO activity decreased by 82 %.

Changes in soluble sugars in relation to desiccation tolerance and effects of dehydration on freezing characteristics of Acer platanoides and Acer pseudoplatanus seeds

Current and previous year needles from three 16 years-old populations of Scots pine (Pinus sylvestris L.) trees were seasonally collected at the three experimental areas: Luboń- close to the phosphate fertiliser factory, Głogów — close to the copper foundry and Kórnik — control site. Głogów is the most polluted site, where at 1998 monthly mean daily concentrations of different pollutants were: SO2 - 17 µg·m−3, NOx - 12 µg·m−3 and dust containing heavy metals as Cu, Pb, Cd - 29 µg·m−3. Trees growing in Luboń were influenced for many years by high concentration of SO2 and fluor compounds. A few years ago emissions were markedly reduced, but low pH of soil and high concentration of aluminium ions still influence the growth of trees. Seasonal changes of ascorbate and thiol content were observed in each needle class and population, with the maximum in the winter and minimum in the summer. In needles from trees growing on polluted sites higher level of ascorbic acid and thiols comparing to control site was observed. Significant differences appeared in each population of Scots pine growing under higher pollution stress in the Głogów site. In needles from trees growing in Luboń significant differences in ascorbic acid and thiols content were evidently less numerous. Needles from polluted sites in some seasons contained significantly more malondialdehyde (MDA) and those was more frequent in Głogów than in Luboń. The results indicated that in the Głogów site trees are more influenced by pollution stress than in Luboń and the defense reaction measured as an increase of the antioxidant level is more evident.

Reactive species, antioxidants and cold tolerance during deacclimation of Picea abies populations

Abstract Using differential thermal analysis (DTA) it was shown that actively growing or developing buds and shoots of Norway spruce die as a result of a single freezing of tissue water, which takes place at temperatures between −8 and −16°C. On the other hand, in winter-hardened organs, tissue water freezes in two successive stages, each of which preceded by supercooling of the tissue water. At first, there is freezing of supercooled water in the extracellular spaces and then there occurs the crystallization by deep supercooling of the remaining tissue water; in winter-resting buds this occurs in the range from −17 to −27°C. Dead or dehardened buds do not demonstrate the tendency towards deep supercooling. The role of water content and of barriers restricting water movement from cytoplasm to the extracellular ice are discussed.

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

The involvement of enzymic antioxidant system, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase in defense reaction to environmental stress evoked by air and soil pollution, was seasonally studied on three populations of Scots pine (Pinus sylvestris L.) growing on experimental areas close two industrial objects in Poland. The first of them (Luboón) is localised near a phosphate fertiliser factory, the second (Głogów) near a copper foundry, and control stand is placed in Kórnik. Głogów is the most polluted site, where in 1998 monthly mean daily concentrations was: SO2 - 17 µg·m−3, NOx- 12 µg·m−3 and dust containing heavy metals (Cu, Pb, Cd) - 29 µg·m−3. Trees in Luboń were influenced for many years by high concentration of SO2 and fluor compounds. Few years ago emissions were markedly reduced, but changes in the soil (low pH and high concentration of aluminium ions) still influence the growth of trees. In needles of two populations: 3 (Russia) and 8 (Poland), from the polluted sites Głogów and Luboń, activities of superoxide dismutase (SOD) and guaiacol peroxidase (PO) were significantly higher compared to Kórnik. However, in one population (16 - Slovakia), such dependance was not evident. Activity of ascorbate peroxidase (AP) measured in winter was also higher in needles from polluted sites. The results indicated that the sensitivity of free radical scavenging system in Scots pine needles differs among populations.