Elżbieta Zenkteler

Cytological analysis of hybrid embryos of intergeneric crosses between Salix viminalis and Populus species.

Intergeneric hybridisation between Salix viminalis L. as the female and four Populus species (Populus trichocarpa, P. tremula, P. × canadensis and P. simonii) as male pollen donors was performed by in vitro stigma pollination. To overcome postzygotic barriers, transfer of hybrid embryos to new medium is necessary. We carried out detailed ultrastructural analyses to establish: (i) at which stage of embryo development the first signs of programmed cell death (PCD) could be detected; and (ii) at which stage the lack of serious or irreversible changes guaranteed that advanced development of hybrid plants could occur after embryo rescue. Transmission electron microscopy and confocal laser scanning microscopy revealed the presence of both developing and degenerating embryos. Developing globular, heart-shaped, and early cotyledonary embryos contained cells of correct ultrastructure. The only sign of intergeneric hybridisation was a delay in development for a few days, in comparison with control embryos. The earliest indicators of embryo degeneration were noted at 9 days after pollination (DAP). The most common indicators were excessive embryo vacuolisation, which was characterised by a large number of vesicles and formation of small vacuoles, as well as enlarged central vacuoles. Extended plastid thylakoids, folding of the cell wall, and autophagosomes were observed. Our detailed investigation of PCD in hybrid embryos enabled us to conclude that the embryo rescue technique was most effective in intergeneric willow × poplar crosses if applied between 9 and 16 DAP.

Phenolic compound localisation in Polypodium vulgare L. rhizomes after mannitol-induced dehydration and controlled desiccation.

Polypodium vulgare L. is a desiccation-tolerant fern that can withstand successive dry periods in its life cycle. To better understand this mechanism, the current study was undertaken to assess the role of phenolic compounds in rhizome dehydration and determine their localisation in the rhizome cells after enforced dehydration in mannitol solution or controlled desiccation with or without abscisic acid (ABA) pretreatment. Phenolic distribution at the subcellular level was studied using gold particle-complexed laccase. Cells from different tissues: cortical parenchyma, endodermis and stelar elements—pericycle, sieve cells and vascular parenchyma were observed under a transmission electron microscope (TEM). The content of phenolic compounds was greater in ABA-untreated rhizomes after enforced dehydration in mannitol solution and subsequent rehydration. After controlled desiccation the phenolic content significantly increased in ABA-untreated rhizomes. A large number of phenolic compound deposits were present in all types of rhizomatous cells. Phenolics were widely distributed in the vacuoles of all cells, and in the secondary cell walls of sieve cells, although scattered labelling was hardly ever observed in the primary cell walls. In dehydrated and plasmolysed cells from the cortex and endodermis, phenolic compounds were present in the apoplastic compartments between the plasma membranes and the cell walls. There is evidence that abscisic acid plays a role as a crucial antioxidant resulting in no damage and a lower level of phenolic increase as compared to ABA-untreated rhizomes. Moreover, the location of phenolics suggests a protective chemical barrier against environmental stresses.

Influence of the biocides PPMtm and Vitrofural on bacteria isolated from contaminated plant tissue cultures and on plant microshoots grown on various media

Summary Bacterial 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 concentration of biocide, and the number of days from the start of the assay. PPM™ inhibited the growth of M. lusitanum for 21 d, P. putida for 14 d, S. pasteuri for 3 d, and Paenibacillus spp. and S. marcescens for 2 d. Vitrofural limited the growth of M. lusitanum for 21 d, P. putida for 14 d, Paenibacillus spp. for 7 d, and S. marcescens for 1 d. PPM™ at 3 ml l–1, 5 ml l–1,or 10 ml l–1, and Vitrofural at 25 mg l–1, 35 mg l–1, or 45 mg l–1 significantly affected the multiplication and rooting characteristics of microshoots. The influence of each biocide depended on its concentration, the plant genotype, and the type of medium used. In most cases, both biocides decreased shoot and root lengths and shoot and root numbers, compared to the untreated controls. Both biocides also showed some positive effects on selected plant genotypes. At all concentrations, PPM™ increased the number of axillary shoots in anthurium, while all concentrations of Vitrofural increased the number of axillary shoots in blackberry. Although PPM™ and Vitrofural decreased shoot multiplication and some rooting characteristics, if necessary they may be added to plant tissue culture media. However, their toxicity towards explants of a given plant genotype should first be tested.