Stratification, Scarification and Application of Phytohormones Promote Dormancy Breaking and Germination of Pelleted Scots Pine (Pinus sylvestris L.) Seeds

Funding: This work was from a Subvention of the Ministry of Science and Higher Education in Poland SUB/2019-0419 000 000-D404.Peer reviewedPublisher PD

The Impact of Biotic and Abiotic Stress Factors on Development of European Ash Tissue Cultures

Fraxinus excelsior L. is threatened by a variety of environmental factors causing a decline of the species. The most important biotic factors negatively affecting the condition of the F. excelsior population are fungi such as the pathogen Hymenoscyphus fraxineus. Abiotic factors with potentially harmful effect to the F. excelsior population are the accumulation of heavy metals and salinity in soils. Thus, the aim of this study was to investigate the impact of selected biotic and abiotic stress factors to determine which of them pose a threat to European ash. The study was conducted using in vitro techniques based on callus and seedlings regenerated via indirect organogenesis. Tissue cultures exclude the influence of other factors, including the environmental impact on ash extinction. The results confirmed very strong pathogenic potential of H. fraxineus in which after 14 days the callus tissue cells died as the tissue failed to activate its defense mechanisms. Experiments showed the high toxicity of cadmium in concentration of 0.027 mmol/L. Salinity caused the activity of oxidation enzymes to vary among seedlings and calluses in the control suggesting the enzymes play a role in controlling the morphogenetic development of tissue cultures

Response of the callus cells of fir (Abies nordmanniana) to in vitro heavy metal stress

The aim of the presented research was to investigate the effect of three heavy metals - lead, cadmium and copper - on the callus cells of Abies nordmanniana. The toxicity degree and toxicity effect of the selected heavy metals was determined on the embryonic level. On the basis of the spectrometric analyses as well as macroscopic and microscopic observations, this research referred to the accumulation of heavy metals in tissues, assuming that this mechanism is related to the acquisition of tolerance by cells exposed to this type of abiotic stress. Moreover, the effect of the genotype of fir on the cell defence, that is, the induction of tolerance, was analysed. Understanding of the issues related to the heavy metal resistance of plant genotypes in future may contribute to the selection of genotypes of individuals that are more resistant to stress factors, particularly in the multi-directional and rational forest management. The results showed that lead (20 mg l-1), which proved to be the most toxic amongst the three examined heavy metals, has the most severe negative effects on the tissue of fir trees. Copper (20 mg l-1) was accumulated for a long time in the cells of fir trees, and it was not degraded or excreted outside the tissues even after three weeks of in vitro culture. Of the three tested genotypes, G14 had the greatest tendency to accumulate each of the examined metals, that is, it appeared to be the least tolerant genotype

The use of callus tissue in forest tree biotechnology: studies in vitro

Kalus występujący w warunkach ex vitro jako tkanka przyranna jest obiektem badaniach biotechnologicznych in vitro takich jak: uzyskiwanie metabolitów wtórnych, analiza patogeniczności na poziomie embrionalnym, krioprezerwacja materiału roślinnego, uzyskiwanie roślin transformowanych i analiza wpływu metali ciężkich na rośliny. Kalus otrzymuje się m.in. metodą somatycznej embriogenezy i organogenezy in vitro jako jej pośredni efekt. Istnieje duże zainteresowanie otrzymaniem kalusa drzew leśnych. Kalus roślin drzewiastych zaindukowany w warunkach in vitro pod wpływem elicitorów w postaci grzybów produkuje metabolity wtórne: monoterpeny i izoproteidy z Betula sp. oraz glikozydy fenolowe u rodziny Salicaceae. Substancje te znalazły zastosowanie nie tylko w przemyśle farmaceutycznym i kosmetycznym, ale również mają silne działanie odstraszające zwierzęta roślinożerne. Kultury dualne in vitro wykazały, że kalus nieembriogenny poddany wpływowi czynników stresowych (grzyb patogeniczny) wytwarza białka odpornościowe o niskiej masie cząsteczkowej. Daje to podstawę do oceny stopnia patogeniczności grzyba na poziomie embrionalnym. Krioprezerwacja kalusa embriogennego znajduje szerokie zastosowanie w rozszerzaniu banku genów. Badano także wpływ metali ciężkich na kalus.Callus, occurring ex vitro as wound tissue, is a subject of extensive biotechnological in vitro research, including: obtainment of secondary metabolites, pathogenicity studies at embryonic level, cryopreservation of plant material, obtainment of transformed plants and influence of heavy metals on plants. Callus can be obtained, among others, as an indirect effect of somatic embryogenesis and organogenesis in vitro. Hence, there is considerable interest in application of this method in forest trees and in callus obtained as a result thereof. Callus of woody plants, induced in vitro under the effect of elicitors in the form of fungi, produces secondary metabolites, such as: monoterpenes and isoprenoids in Betula sp. and phenolic glycosides in the family Salicaceae. These secondary metabolites have been used not only in the pharmaceutical and cosmetic industries, but also as strong repelling agents against herbivores. On the other hand, studies in dual in vitro cultures have shown that non-embryogenic callus of e.g. Pinus sylvestris subjected to a stress factor such as pathogenic fungus, produces in response low molecular weight proteins classified as PR-type (pathogenesis related) immune proteins. These observations give the basis for the assessment of pathogenicity and the level of threat posed by the fungus at the cellular level. Also, cryopreservation of embryogenic callus is widely used, among others, in gene bank reservoirs of valuable plant genotypes. Biotechnological studies involving callus of different forest tree species complement and support the development of disciplines such as physiology, phytopathology or plant breeding and selection

Secondary Metabolites Produced by Trees and Fungi: Achievements So Far and Challenges Remaining

Secondary metabolites are ubiquitous substances occurring naturally in trees and microorganisms. They are produced in various metabolic pathways which determine their structure and biochemical proprieties. However, the biological functions of many secondary metabolites remain undetermined. Usually, the amounts of secondary metabolites produced by trees under natural conditions are limited, which makes their mass production difficult and not cost-effective. Metabolites occurring naturally in plants, including gymnosperm and angiosperm trees, as well as in fungi, are important biologically active substances used by many industries and in modern medicine. The huge variability and potential of biological activity present in secondary metabolites make it possible to replace most of them with compounds of completely natural origin. The current breakdown of metabolites, together with the most important examples of compounds and their uses, are presented in this overview. The possibility of increasing the number of secondary metabolites in a specific environment through interaction with the most known biotic factors is discussed. The use of in vitro culture for the production of secondary metabolites and their extraction, as well as the possibility of subsequent analysis, are described. The current literature on the metabolites produced by individual species is presented

Transformation of European ash (Fraxinus excelsior L.) callus as a starting point for understanding the molecular basis of ash dieback

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity

Investigation of the biocontrol potential of two ash endophytes against Hymenoscyphus fraxineus using in vitro plant-fungus dual cultures

Development of effective biocontrol procedures using ash endophytes to combat an ash pathogen Hymenoscyphus fraxineus would be an appropriate contribution to the ongoing effort to protect European ash stands against ash decline. In this study we investigated the biocontrol potential of two ash endophytes, Thielavia basicola and Minimidochium sp., against H. fraxineus using in vitro plant-fungus and fungus-fungus dual cultures approach in three biocontrol models. The tests aimed to determine whether the endophytes show antagonism toward Fraxinus excelsior and F. pennsylvanica, to assess the level of antagonism of the endophytes toward H. fraxineus and to identify potential secondary metabolites induced by the presence of H. fraxineus. The results that dual culture experiments modeled according to our design may be a very useful tool to precisely study biocontrol potential of fungi, i.e., without the impact of environmental factors. Such experiments also enable the selection of most resistant ash genotypes and rapid propagation, producing large numbers of pathogen-free seedlings. It should be noted, however, that both of the endophytes tested in the dual cultures strongly inhibited the growth of H. fraxineus. Their growth under the influence of callus/seedlings was also inhibited. Comparison of HPLC profiles showed that the presence of H. fraxineus in the post-culture medium induced the production of an unknown secondary metabolite in this species. Such results suggest that some of the plant–fungus combinations examined in this study may have potential to be developed as biocontrol methods, thus increasing the survivability of ash stands under natural conditions