A study was conducted to investigate the effect of different storage periods and temperatures on pollen viability in vitro and in vivo in plum genotypes ‘Valerija’, ‘Čačanska Lepotica’ and ‘Valjevka’. In vitro pollen viability was tested at day 0 (fresh dry pollen) and after 3, 6, 9 and 12 months of storage at four different temperatures (4, −20, −80 and −196 °C), and in vivo after 12 months of storage at distinct temperatures. In vitro germination and fluorescein diacetate (FDA) staining methods were used to test pollen viability, while aniline blue staining was used for observing in vivo pollen tube growth. Fresh pollen germination and viability ranged from 42.35 to 63.79% (‘Valjevka’ and ‘Čačanska Lepotica’, respectively) and 54.58 to 62.15%, (‘Valjevka’ and ‘Valerija’, respectively). With storage at 4 °C, pollen viability and germination decreased over the period, with the lowest value after 12 months of storage. Pollen germination and viability for the other storage temperatures (−20, −80 and −196 °C) were higher than 30% by the end of the 12 months. Pollination using pollen stored at 4 °C showed that pollen tube growth mostly ended in the lower part of the style. With the other storage temperatures, pollen tube growth was similar, ranging between 50 and 100% of the pistils with pollen tubes penetrated into the nucellus of the ovule in the genotype ‘Čačanska Lepotica’. The results of these findings will have implications for plum pollen breeding and conservation.

In vitro strategies for the propagation and conservation of indigenous species contribute to the sustainable use of plant diversity and are essential for breeding programs as well. In this study, we established an efficient protocol for the micropropagation of autochthonous plum 'Crvena Ranka' and examined the survival and regrowth capacity of in vitro shoots after 3, 6 and 9 months of cold storage (CS) at +5 oC in total darkness. Aseptic culture was established on the Murashige and Skoog medium containing 2 mg l-1 BA, 0.5 mg l-1 IBA and 0.1 mg l-1 GA3 (leaf rosette initiation being 68.8%). During in vitro propagation on the medium of constant hormonal composition, a significant increase in the multiplication index was observed in the third subculture, whereupon it was mainly stable until the fifth subculture. The effect of BA concentration and/or type of auxins (IBA or NAA) on multiplication parameters, as well as on fresh and dry weights of shoots was evaluated. BA at 1 mg l-1 in combination with NAA significantly increased shoot multiplication parameters. The effect of auxins on rooting parameters was monitored as well. Shoots cultured on the medium supplemented with NAA also displayed higher rooting ability (60%), in comparison with those grown on the medium containing IBA at the same concentration (20%). In vitro shoots can be conserved over the medium term under CS conditions up to six months. High survival was achieved after three (94%) and six months (82.5%), while severe signs of necrosis (100%) were noticed after nine months of conservation. Shoots subcultured under standard growth conditions after CS promptly regained their morphology although their capacity for multiplication and rooting was slightly lower than that of non-cold-stored shoots.