Radosav Cerović

Relationship between the concentration of macroelements, their uptake and multiplication of cherry rootstock Gisela 5 in vitro

Sweet cherry rootstock Gisela 5 was micropropagated on Murashige and Skoog (MS) medium, on MS medium containing double-strength macro salts (MS 2×), 1/2 strength (MS 1/2) and 1/4 strength (MS 1/4) with 4.4 μM BA, 0.5 μM NAA, and 0.3 μM GA3. All the analyses and parameters were monitored on day 0, day 20 and day 40 after subculturing. As fresh and dry weight of the explants increased during subculturing, the fresh and dry weight of the media decreased. The pH of the media declined during subculturing following by slow increase on media MS 1/2 and MS 1/4. Gisela 5 showed the best growth and development on MS 2× and MS media with the highest N and P uptake. Growth and multiplication depend on the uptake of these elements from the medium.

Senescence of ovules at different temperatures and their effect on the behaviour of pollen tubes in sour cherry

Abstract The ageing process of the ovules in sour cherry cultivar ‘Cacanski Rubin’ was studied on the basis of the occurrence of fluorescence at five constant temperatures (5, 10, 15, 20 and 25°C) under laboratory conditions for 2 years. A correlation was found to exist between the velocity of ovule senescence and temperature, and the number of days after anthesis. Also, unusual behaviour of pollen tubes was observed in different parts of the ovary, possibly a result of the loss of ovule viability. These phenomena were pronounced in both study years, with only slight differences.

Changes in macroelement content of the media and in sweet cherry Inmil GM 9 shoots during in vitro culture

Summary The paper presents an interrelationship between macroelement content in the culture medium, their uptake from the medium, the content in the explants, as well as multiplication and the in vitro growth of the new rootstock for sweet cherry Inmil GM 9. Cultures were grown on Murashige and Skoog (MS) medium, on MS medium with double-strength, half-strength and quarter-strength macro salts with 4.4.µM BA, 0.5.µM NAA, and 0.3.µM GA3. All chemical analyses and multiplication parameters were monitored on day 0, day 20 and day 40 of culturing. As fresh and dry weight of the explants increased during subculturing, the fresh and dry weight of the media decreased. The pH of the media declined during subculturing, followed by an increase, particularly on MS 1/2 and MS 1/4 media. The genotype Inmil GM 9 showed the best growth and developmenton MS medium, where the N and P uptake was highest. The highest uptake of ions by shoots was recorded on the media with reduced macroelement content, with the exception of phosphorus. The shoots had the highest content of macroelements on MS, except for N which was highest in the explants grown on MS 2x. Leaves are mainsites of macroelement accumulation.

Micropropagation in vitro of highbush blueberry (Vaccinium corymbosum L.)

Murashige and Skoog medium (MS) and modified Anderson’s Rhododendron medium (mAN) were compared for in vitro shoot multiplication of three highbush blueberries ‘Berkeley’, ‘Bluecrop’ and ‘Goldtraube’. All media contained 0.5 mg l−1 zeatin applied either alone or combined with 0.1, 1 and 5 mg l−1 IBA. In vitro rooting was induced using mAN medium supplemented with 0.8 mg l−1 IBA and 4 g l−1 activated charcoal. The results obtained showed that mAN medium is more suitable for in vitro multiplication of the selected highbush blueberry cultivars than MS medium. Low concentration of IBA (≤1 mg l−1) added in zeatinsupplemented mAN medium increases shoot multiplication efficiency of highbush blueberries in vitro and can be recommended for large-scale propagation of high-quality plants. MS medium induced partial or full necrosis of stems and leaves, which was more pronounced on media containing zeatin combined with increasing concentration of IBA. Rooting capacity of shoots varied widely among the tested blueberry cultivars. The highest rooting and acclimatization rates were achieved in ‘Goldtraube’ (82.8% and 91.8% respectively), and the lowest (10% and 66.7% respectively) were in ‘Berkeley’.

Cryopreservation of cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens) shoot tips by droplet-vitrification technique

ABSTRACT The droplet-vitrification technique was applied to in vitro shoot tips of cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens). Explants were precultured in the dark at 23 °C, in liquid MS medium with a progressively increasing sucrose concentration (0.3 M for 15 h, then 0.7 M for 5 h). Loading involved a 30 min incubation of explants in a solution comprising 1.9 M glycerol and 0.5 M sucrose. Explants were dehydrated at room temperature using a solution PVS A3 [Murashige and Skoog (MS) liquid medium, 22.5% (w/v) sucrose, 37.5% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide] for 30, 40 and 50 min and the PVS3 solution [MS liquid medium, 50% (w/v) sucrose, 50% (w/v) glycerol] for 60, 90 and 120 min. Explants were cooled by direct immersion in liquid nitrogen (LN) in 10 μl droplets of vitrification solution placed on aluminum foil strips. The foil strips were retrieved from LN and immersed in preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, and an equal volume of unloading solution at room temperature was added for further incubation for 30 min. Shoot tips were transferred onto the regrowth medium, cultivated in the dark for 7 days before being incubated under standard conditions. Three weeks after transferring the shoot tips onto the regrowth medium, the survival rate of control and cryopreserved explants of Gisela 5 dehydrated with PVS A3 was 100%, regardless of the treatment duration. After dehydration with solution PVS3, the survival varied between 70 and 100% for control explants and 78 and 95% for cryopreserved shoot tips. Gisela 5 shoot tips dehydrated for 40 min with PVS A3 vitrification solution demonstrated the best regrowth (38%). When using the PVS3 solution, survival of cryopreserved shoot tips was the highest (95%) after 60 min treatment followed by 40% regrowth. After three successive subcultures on shoot multiplication, medium shoots recovered viability, multiplication ability and morphology equal of that prior to cryopreservation.

The effect of genotype and temperature on pollen tube growth and fertilization in sweet cherry (Prunus avium L.)

The study of pollen performance in vivo of four self-incompatible sweet cherry cultivars—‘Karina’, ‘Kordia’, ‘Regina’ and ‘Summit’ was performed. The results were considered in the context of climatic conditions of West Serbia, bearing in mind increased frequency of seasons with higher temperatures during the flowering. Each cultivar was used as polleniser and pollinated cultivar, and together with the open pollination variant, a total of 16 combinations were analysed during the three-year period. The effect of variability factors on pollen tube growth efficiency occurred as a common impact of polleniser, its requirement to flowering temperatures, the influence of pollinated cultivar on pollen performance, and the response of male–female relations to air temperature. ‘Summit’ induced the highest pollen tube number, while the pollen tube number of ‘Kordia’ was the lowest. Higher temperatures during the flowering have caused a decrease in the number of pollen tubes in ‘Kordia’ and ‘Karina’, and increase of their number in ‘Regina’ and ‘Summit’ as pollenisers. The genotype-dependent response of pollenisers in terms of air temperature during the flowering has not consistently manifested, and was partially modified by the influence of the pollinated cultivar. The main tendencies were more apparent in the ‘borderline cases’, i.e. in pollination of cultivars having diametrically opposite requirements, than in those of cultivars having similar requirements regarding the flowering temperature.

Cryopreservation of Serbian autochthonous Prunus spp. by droplet-vitrification

Abstract In vitro shoot tips of Serbian autochthonous plums ‘Sitnica’ (Prunus domestica L.) and ‘Crvena Ranka’ (Prunus insititia L.) were tested for recovery after cryopreservation using the droplet-vitrification technique. After 30-min loading with 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated at room temperature for 10, 20, 30, 40 and 50 min with PVS A3 solution (37.5% glycerol, 15% dimethylsulfoxide, 15% ethylene glycol and 22.5% sucrose) or for 60, 90 and 120 min using PVS3 solution (50% glycerol and 50% sucrose). Rewarming was performed in unloading solution containing 0.8 M sucrose for 30 or 60 min. Duration of PVS3 treatment significantly affected survival (27.3-72.7%) and regrowth (0-18.2%) of cryopreserved explants in plum ‘Sitnica’, with the highest values of both parameters being achieved with the 90-min treatment. Also, survival of explants dehydrated with PVS A3 solution significantly varied between 18.2-73.9%, depending on duration of both dehydration and unloading treatments. However, cryopreserved explants displayed a very low regrowth capacity, the highest being 10% for 30-min dehydration in combination with 60-min unloading. ‘Crvena Ranka’ exhibited a higher regrowth capacity after cryopreservation. Duration of PVS3 treatment significantly affected survival (22.2-77.8%) and regrowth (0-30.0%) of cryopreserved explants, with the highest values of both parameters being achieved with the shortest treatment duration. As regards PVS A3 treatments, both survival and regrowth significantly varied between 27.3- 81.8%, and 0-36.4%, respectively. The highest regrowth was achieved with 20- and 30-min treatment durations combined with 30-min unloading. Further optimization of the protocol is necessary to improve recovery after cryopreservation.

Dynamics of pollen tube growth and embryo sac development in Pozna Plava plum cultivar related to fruit set

A newly released, late ripening plum cultivar Pozna Plava sets fruit poorly, although it produces high quality fruit. This study aimed to evaluate which factors in the reproductive process could be related to the lack of fruit set. In two consecutive years, establishment of a suitable polleniser and the stage of ovule development at anthesis as well as initial and final fruit set have been studied. In addition to this, the impact made by temperature fluctuations on the interaction between male gametophytes and female sporophytes was also analysed. Growth of the pollen tubes in the style and penetration into the nucellus as well as fruit set were more effective in cross-pollination than in open and self-pollination. A relative delay in ovule development was observed, and most ovules had an embryo sac with eight nuclei. Considering the results of the quantitiative parameter study of pollen tube growth in the ovary as well as the results of the stage of ovule development, a conclusion can be made that this cultivar is characterised by an extremely short effective period of pollination.

Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

Summary In vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.