In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Bacteria in the plant tissue culture environment

Bacteria and plants are joined in various symbiotic relationships that have developed over millennia and have influenced the evolution of both groups. Bacteria inhabit the surfaces of most plants and are also present inside many plant organs. These bacteria may have positive, neutral or negative impacts on their plant hosts. Probiotic effects may improve plant nutrition or increase resistance to biotic and abiotic stresses. Conversely pathogenic bacteria may kill or reduce the vigor of plant hosts. In addition some bacteria inhabit plants and profit from excess metabolites or shelter while not injuring the plant. Micropropagation of plants is based on the stimulation of organogenesis or embryogenesis from explants that are superficially decontaminated and placed into a sterile environment. If successful, this process removes bacteria from surfaces, but those inhabiting inner tissues and organs are usually not affected by these steriliants. In vitro conditions are designed for optimal plant growth and development, however these conditions are also often ideal for bacterial multiplication. The presence of bacteria in the in vitro environment was almost universally considered negative for plant culture, but more recently this view has been questioned. Certain bacteria appear to have a beneficial effect on the explants in culture; increasing multiplication and rooting, increasing explant quality, and organo- and embryogenesis of recalcitrant genotypes. The most important role of beneficial bacteria for micropropagated plants is likely to be during acclimatization, when growth is resumed under natural conditions. This review includes the role of bacterial interactions in plants, especially those grown in vitro.     

Detection and identification of Phytophthora alni

In 2004 Brasier et al. described new species--Phytophthora alni, which was especially aeggressive to alder. Now, this Phytophthora disease of alder is widely distributed in Europe as well as in Poland. In this research note we report on identification and detection of P. alni from water and soil samples using PCR method with species-specific primers. Dilution series of P. alni zoospore were used to test the potential sensitivity of the PCR detection methods. Zoospores of P. alni were produced by flooding of 1-week-old Frozen Pea Medium (FPM) cultures in Petri dishes with 30 ml distilled water. The dishes were incubated at 20 degrees C. After 5 days, sporangial production was checked using a binocular microscope and plates were placed at 4 degrees C for 1 h to enhance zoospore release. Zoospores were counted under the microscope using Burker's cabin. A dilution series of zoospores ranging from 5 to 5000 per 200 microl was prepared in autoclaved distilled water and in 1 g samples of autoclaved soil. DNA was extracted from artificially infected water and soil, and purified using the CleanUp Kit (A&A Biotechnology). Zoospores of P. alni in the water were detected by PCR in 5 x 10(3), 5 x 10(2), 5 x 10(1) concentrations. In case of detecting spores in the artificially infected soil it succeeded only for two highest concentrations, i.e. 5 x 10(3), 5 x 10(2) and only when the DNA was additionally purified.     

The influence of the cooling of donor cultures on the in vitro adventitious regeneration and carbohydrate metabolism of four dwarfing apple rootstocks

We examined the effects of cooling applied for 4 to 20 weeks on donor cultures of four dwarfing apple rootstocks (P16, P22, P59 and M26). Our aim includes increasing their competence for in vitro adventitious shoot regeneration from the leaves. Donor cultures were maintained on a shoot multiplication medium at 4°C in the dark for 4 months, followed by subculture on a fresh medium for 4 weeks. The cooling of the cultures caused an increase in the adventitious shoot number and a decrease in the starch content and an increase in the soluble sugar content (monosaccharides, raffinose and stachyose). The accumulation of stachyose in response to cold is a new observation, and it suggests that raffinose and stachyose play important role in the acclimation of dwarf apple rootstocks to low temperatures.     

Effects of mineral composition and acidity of media,saccharose level,brand and quantity of agar on rooting of fruit rootstocksin vitro

The influence of the mineral composition and acidity of media, saccharose level, brand and quantity of agar onin vitro shoot rooting of P 60 and P 2 apple and quince S 1 root-stocks were compared. Among the tested salt compositions (full WMP and 1/2 WMP, full MS and 1/2 MS) the most suitable for rooting was composition of WMP. Further modifications in quantity of nitrogen, H₃BO₃ CaCl₂ and MgSO₄ of WMP medium did not have a positive effect on rooting. 30 g l^?1 saccharose gave better results than 20 g l^?1. Acidity of WMP medium tested in a pH range of 4.0 to 6.5 did not affect rooting of P 60 rootstock but media adjusted to pH 4.5 and 5.0 were better for rooting of recalcitrant P 2 rootstock. Among 4 agar brands (Difco Bacto, Oxoid No.1, Japanese commercial fibre and Japanese commercial powder) tested at 6 g l^?1, the most effective was the last one, but its quantity had no effect in the range of 3 to 10 g l^?1 for rooting of P 60 rootstock.     

Phytophthora spp. associated with nursery reservoirs, rivers and drainage canals

Phytophthora spp. were recovered from water using rhododendron leaves as baits for detection of that group of organisms. They were was found in 4 rivers, 2 hardy nursery water reservoirs and nursery drainage canal from May to October, 2006. Analysis of spots number on rhododendron leaf baits as the measure of Phytophthora spp. density showed that place of baits holding had significant influence on the species occurrence. Significantly more spots, especially in July surveying, were observed on baits holding in Skierniewka and Zwierzynka rivers swimming through agriculture and forest area than in Ner, the river of horticulture area. In nursery water containers and drainage canal higher Phytophthora density was noticed on August than other periods of surveying.     

Determination by GISH and FISH of hybrid status in Lilium

In the genus Lilium, plants obtained from crosses, especially between distant relatives, are not always hybrids because embryos can develop as a result of apomixis. These plants constitute genetic material of the maternal parent only. In this study, verification of hybrid status of plants which have been obtained from the crosses 'Marco Polo'xLilium henryi and 'Expression'xL. henryi was performed through the use of cytological and molecular cytogenetic methods. According to cytological analyses, all genotypes tested had 2n = 2x = 24 chromosomes. Genomic in situ hybridisation (GISH) was used for hybrid verification. In hybrid plants, this method distinguished all paternal and maternal chromosomes at the stage of somatic metaphase and prophase. For GISH, paternal genomic DNA was used as a probe and maternal DNAs were used as blocks. Fluorescence in situ hybridisation (FISH) with 5S rDNA and 25S rDNA probes was used as the second method of hybrid verification. Selected chromosome markers based on genome-specific localisation of rDNA loci were used for analysis of the F1 hybrids obtained from the crosses 'Marco Polo'xL. henryi and 'Expression'xL. henryi. The presence of marker chromosomes characteristic for each of the paternal genotypes was a confirmation that the plants obtained were hybrids.