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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.

Among the numerous protocols that describe the extraction of DNA, those relating to the isolation of DNA from infected plants, are rare. This study describes a rapid and reliable method of extracting a high quality and quantity of DNA from rhododendron leaves artificially infected with Phytophthora cactorum, P. cambivora, P. cinnamomi, P. citrophthora, and P. plurivora. The use of the modified Doyle and Doyle protocol (1987) allowed us to obtain high quantity and quality DNA (18.26 μg from 100 mg of the fresh weight of infected leaves at the ratios of A260/280and A260/230- 1.83 and 1.72, respectively), suitable for conventional polymerase chain reaction (PCR) and real-time PCR amplifications.

The strains of bacteria Paenibacillus glucanolyticus, Curtobacterium pusillum and Methylobacterium extorquens were isolated as non-deleterious contaminations from hosta or raspberry tissue cultures. Microshoots of chrysanthemum, gerbera, hosta and rose were inoculated with these bacteria and their influence on shoot multiplication and rooting was evaluated. None of the investigated bacteria caused symptoms of hypersensitivity or vitropathy on the shoot explants at rooting and shoots multiplication. C. pusillum stimulated axillary shoot formation in all studied plant genotypes. Length and number of rose roots and their length were higher but number of roots and their length in chrysanthemum were lower in inoculated than in controls. The number and the length of shoots and roots in gerbera and hosta and the number of shoots in chrysanthemum inoculated with M. extorquens were higher but shoot length of chrysanthemum and rose, and root length of rose were lower as compared with controls. P. glucanolyticus influenced higher number and length of chrysanthemum shoots and root length of chrysanthemum and gerbera than noninoculated control but the number of gerbera and hosta roots was lower and root length of rose was as low as 0.2 cm. All assessed bacteria were able to assimilate atmospheric nitrogen and M. extorquens and P. glucanolyticus were able to produce IAA.

SummaryBacterial contamination is often a serious problem during plant micropropagation. When disinfection of the initial explants fails, and bacteria are not detected at the initial stage of propagation, they can survive unobserved as a contamination in the plant explants and only appear when the population of microshoots is large. Biocides added to the culture media can be used to help reduce bacterial multiplication. This research was aimed at determining whether the use of PPM™ and Vitrofural could restrict the growth of various bacteria (Methylobacterium lusitanum, Paenibacillus spp., Pseudomonas putida, Serratia marcescens, and Staphylococcus pasteuri) isolated from plant cultures, and whether these biocides were detrimental to shoot multiplication and rooting in anthurium, blackberry, chrysanthemum, hosta, raspberry, and strawberry microshoots. PPM™ and Vitrofural restricted the growth of bacteria in agar-diffusion assays for periods of 1 d to 21 d, depending on the bacterial genotype, the type and...

First Report of Phytophthora Inflata in Polish Rhododendron Nursery Phytophthora inflata Caros & Tucker was detected together with P. citricola Sawada from rhododendron cv. University of Helsinki with necrotic spots on leaves. Spots were enlarged on tip parts of the stems. The rhododendron plantswere taken from hardy ornamental stock. Inoculation of the leaf blades of 15 species and cultivars of rhododendron with P. inflata and P. citricola resulted in the development of necroses on all of them. Reaction of tested rhododendron species and cultivars for P. inflata and P. citricola was similar. The pathogen also colonized leaf blades of Goultheria decumbens, Kalmia ostroseptice, Vaccinium corymbiferum and V. vitis-idaea. The quickest spread of necroses was observed on K. ostroseptice and V. corymbiferum. In greenhouse trials P. citricola and P. citrophthora caused dieback of more rhododendron plants than P. inflata.

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.

Rose genotypes can be recalcitrant in in vitro regeneration. A lack of competence for regeneration results in unsatisfactory numbers of adventitious shoots, which diminishes achievements in use of genetic engineering in rose modern breeding. Among the factors which can partially overcome this problem is the use of thidiazuron (TDZ) in the induction phase of regeneration. We studied the use of TDZ in concentrations 0.05 and 1.0 mg l−1, in the medium for the culture of donor shoots with the aim of increasing the regeneration competence of five rose cultivars differing in their regeneration ability. All the tested genotypes increased their regeneration potential when the donor shoots were cultured on TDZ media for 15 or 30 days. The strongest effect, doubling to quadrupling the number of shoots, was obtained in the cultivars characterized by the lowest regeneration potential. This enhanced regeneration ability was maintained through two or three subsequent 4-week long regeneration passages.

P. lacustris sp. nov. (formerly known as Phytophthora taxon Salixsoil) was first isolated in 1972 in the UK and then in many other European countries, including Poland. The aim of this work was a morphological, physiological, and genetic characterization of the P. lacustris isolates by means of the daily growth rate, mycelium morphology, and generative and vegetative structures, depending on the temperature of incubation and growth media. In addition, the ability to colonize willow shoots and leaves was estimated. Out of 114 isolates of P. lacustris obtained from water habitats located near plant nurseries in central and southeastern Poland in 2007-10 that were identified on the basis of molecular tests which showed high diversity in colony growth patterns and daily growth rates, 10 groups were separated by means of Duncan’s test. Representatives of these 10 groups together with three reference isolates – P. lacustris P245 as the holotype, P. gonapodyides CBS 117380 as a specimen most closely related phenotypically to P. lacustris, and P. cactorum as a positive control of forest trees’ pathogen – were researched. Great heterogeneity in the growth rates and morphology of mycelium, as well as in the structure of zoosporangia and hyphal swellings, were observed. Moreover, the isolates differed in their ability to colonize the willow leaves and shoots in the in vitro tests. Some correlation may be found between the daily growth rates and colonizing abilities, and between the daily growth rate and the dimension of the sporangia. Also, a large genotypic variation between the isolates based on the fingerprint patterns generated by molecular techniques (RAPD and ISSR) was obtained.

We examined the effect of FeEDDHA on multiplication via axillary branching and adventitious shoot regeneration from leaf explants in five red raspberry cultivars. When applied during multiplication, FeEDDHA reduced chlorosis, increased content of chlorophyll and iron but had no effect on the number of side shoots in four of the five cultivars. Addition of FeEDDHA to regeneration medium increased the percentage of regenerating leaves in some cultivars and the number of adventitious shoots in all five cultivars.

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.

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’ � /Lilium henryi and ‘Expression’ � /L. 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’� /L. henryi and ‘Expression’ � /L. henryi . The presence of marker chromosomes characteristic for each of the paternal genotypes was a confirmation that the plants obtained were hybrids.

Plants were obtained by ovule rescue from pollinations of lily cultivars belonging to the group oriental hybrids with pollen of Lilium henryi, L. pumilum and L. x formolongi. In order to develop molecular DNA markers useful for separation of the hybrids all the parental genotypes, resulting seedlings and regenerants were fingerprinted using RAPDs. The products of amplification were detected for all the forty primers used. Eight primers generated reproducible polymorphic bands within the pair 'Marco Polo' and L. henryi, six primers within 'Alma Ata' and L. pumilum, and 'Muscadet' and L.x formolongi. It was found that some bands could serve as markers to detect hybrids at the in vitro stage.

The removal of all developed leaves from 1 to 1.5 cm shoots of Codiaeum variegatum Blume var. pictum Muell. Arg. ‘Excellent’, cultured on the Murashige–Skoog medium containing 4.4 μM (1 mg l−1) 6-benzylamino-purine (BAP) and 5.8 μM (2 mg l−1) gibberellic acid (GA3), doubled the number of axillary shoots in comparison to non-defoliated controls. An additional increase in shoot numbers was achieved when the shoots were placed on the medium horizontally or vertically in an inverted position with the shoot tip down. Shoot tip removal slightly improved axillary branching, and additional defoliation strengthened this effect. Exogenous indole-3-acetic acid (IAA) decreased shoot numbers, and 2,3,5-triiodobenzoic acid (TIBA) did not replace defoliation nor diminish the size of callus formed at the base of the shoots. Almost all the shoots obtained were able to root on the medium with 4.9 μM (1 mg l−1) indole-3-butyric acid (IBA), and acclimatize in the greenhouse.

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars.

The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. ‘Centennial’ were examined. Agrobacterium tumefaciens strain EHA105/p35SGUSInt was more infective than LBA4404/pBI121 as determined by numbers of sectors expressing β-glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by addition of acetosyringone, indicating that a hypersensitive response to bacterial infection may reduce organogenetic potential. Likewise exposure of co-cultivated explants to kanamycin or geneticin in selective media reduced regeneration efficiency. Addition of 500 mg l-1 carbenicillin slightly increased numbers of regenerating shoots. Tranfformed shoots were obtained only when kanamycin selection was initiated 1 or 2 days after co-cultivation. Presence of transgenes in geneticin-resistant shoots was confirmed using polymerase chain reaction and Southern hybridization assays.

L-arginine was added to the rooting media for apple rootstock shoots taken from proliferating cultures. The effect was studied in combination with other rooting factors such as: phloroglucinol, initial dark period, concentration of indol-3-yl butyric acid and inorganic nitrogen levels. In all treatments, arginine caused an increase in root number per rooted shoot and enlargement of the shoot base. Arginine was especially effective with low indol-3-yl butyric acid levels as well as without it, and with low or no inorganic nitrogen. The effect of arginine on root number interacted with dark treatment and with phloroglucinol. The most efficient amount of arginine was 200 mg l−1. The possible influences of arginine on rooting are discussed.

We undertook an analysis of the genomic relationships between 15 isolates of Phytophthora ramorum Werres, de Cock et Man in't Veld, obtained from symptomatic plants growing in Polish ornamental nurseries, and 2 representatives of the European population and 3 of the North American population. Dendrograms were generated by UPGMA based on 786 amplification products obtained in ISSR-PCR reactions. The representatives of the European population and 13 of the "Polish" isolates formed a common cluster. The other 2 "Polish" isolates, which were found in 1998, and the 3 American representatives formed 2 separate clusters. There was no observed link between genomic distance on the basis of polymorphism and the origin of the isolates from plant species.

The youngest leaves, petioles, and roots of micropropagated anthurium explants of cv. Bolero were regenerated in vitro on media consisting of ½ Murashige-Skoog salts with the ammonium nitrate content lowered to 1/4, and with different combinations of growth regulators. In all the experiments, NaCl was included in the medium at a concentration range of 0, 10, 20, 40, 80, 160 and 320 mM, or 0, 25 and 50 mM. The most effective medium for shoot regeneration contained 0.5 mg/L thidiazuron and 0.5 mg/L NAA. The earliest regeneration was from leaves, then from petioles and roots. The highest NaCl concentration that the explants could survive and regenerate in was below 40 mM when BAP and 2,4-D were used, and 50 mM when TDZ and NAA were used. The shoot regeneration was generally preceded by a globular callus that was light green on the medium without NaCl and yellow with scattered green points on the medium with NaCl. The green shoots on the NaCl medium regenerated 2-3 months later than those on the NaCl-free medium. Most of these shoots yellowed and proved to be escapers, but five survived prolonged selection.

The pattern of regeneration from tissues of Paeonia mlokosewitschii and P. tenuifolia cultured in vitro in the same chemical conditions depended on the initial explant. Direct shoot regeneration was obtained from the bases of petioles and petals, and leaf veins. Vegetative initial buds and regenerated in vitro shoots produced on their bases slowly growing nodular callus which was very productive in repetitive shoot regeneration. The tops of stems, flower bases, sepals, petals and ovary walls produced small callus which regenerated white and red spherical structures within 1.5 years. After that time also from those cultures arised nodular, shoot regenerating callus developed.

Phytophthora plurivora was the most often detected species from water using rhododendron baits. The species was isolated from water of two rivers, Jasieniec and Korabiewka, a water pond and a drainage canal from March to November, 2008 (in Korabiewka river also in December). The highest population density of P. plurivora was observed in March and April in water pond and canal, and in May in both analysed rivers. In laboratory trials all tested isolates colonized rhododendron and poplar leaves. Isolates from drainage canal were the most pathogenic for rhododendron. Isolates detected in March from water pond and two rivers caused the quickest spread of necrosis on leaf blades. On poplar leaves the fastest development of necrotic spots was observed when isolates obtained in June and November were used for inoculation, while the isolate from September sample was less pathogenic.