Anna Mikuła

Somatic embryogenesis of Gentiana genus. I. The effect of the preculture treatment and primary explant origin on somatic embryogenesis of Gentiana cruciata [L.], G.pannonica [Scop.], and G.tibetica [King]

Experiments were carried out on three selected species of Gentiana genus: Gentiana cruciata (L.), G. pannonica (Scop.), and G. tibetica (King). Using MS medium supplemented with 2,4-D and kinetin a somatic embryogenesis system of plant regeneration was developed. Induction and intensity of somatic embryogenesis as the effect of integration of the following factors were studied, specifically: seedling pre-treatment (with and without GA3 treatment), light condition (light versus the dark), and type of explant (root, cotyledon and hypocotyl). Numerous significant differences between studied factors were observed and statistically proved.

Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintains (epi)genetic uniformity of regenerants

The embryogenic cell suspension culture of Gentiana cruciata, cryopreserved by the encapsulation/dehydration method, survived both short- (48xa0h) and long-term (1.5xa0years) cryostorage with more than 80% viability. To assess the influence of cryotreatments on the embryogenic potential, a proembryogenic mass was encapsulated and exposed to the following treatments: (1) osmotic dehydration (OD), (2) ODxa0+xa0air desiccation (AD) and (3) ODxa0+xa0ADxa0+xa0cryostorage (LN). The somatic embryogenesis efficiency increased ten times after osmotic dehydration. The AD and LN cryotreatments did not cause any significant alterations in somatic embryo production. We monitored the (epi)genetic stability of 288 regenerants derived from: non-cryotreated, short-term, and long-term cryostored tissue using metAFLP markers and ten primer combinations. Changes in the sequence and DNA methylation levels were studied by subjecting the DNA to digestion with two pairs of isoschisomer restriction enzymes (KpnI/MseI and Acc65I/MseI). Two new AFLP unique DNA fragments at the DNA sequence level, with no differences at the methylation level, were found between regenerants derived from cryopreserved tissue, compared with the non-cryotreated controls. The Acc65I/MseI methylation levels for the three groups of regenerants were not significantly different. Cluster analysis was capable of identifying a number of sub-clusters. Only one of the sub-clusters comprises almost all regenerants derived from non-cryotreated and short-term cryostored tissue. Plantlets derived from long-term cryostored tissue were grouped into separate clusters. The observed AFLP alterations did not appear to be associated with the use of cryopreservation, but were probably related to the process of in vitro culture.

Effect of sucrose concentration on photosynthetic activity of in vitro cultures Gentiana kurroo (Royle) germlings

In this work, the effect of sucrose on photosynthetic activity during in vitro culture was studied. Experiments were carried out using uniform somatic embryo-derived germlings of Gentiana kurroo (Royle) confirmed by chromosome counting and flow cytometry technique. Photosynthetic activity was measured by chlorophyll a fluorescence and gas exchange method. The efficiency of photosynthetic apparatus as measured by the ratio Fv/Fm, Yield and qP (light phase of photosynthesis) was the highest when the medium was supplemented with 0.3% sucrose which well corresponded with plant gas exchange. Taking all data into consideration for the best development of photosynthetic apparatus and the most efficient of net photosynthesis of studied germlings would be medium supplemented with 0.2–0.4% of sucrose.

Somatic embryogenesis of Gentiana genus. III. Characterization of three-year-old embryogenic suspensions of G.pannonica originated from various seedling explants

Experiments were carried out with three-year-old embryogenic suspension culture of Gentiana pannonica Scop. The initial explant for the suspension determinated both the embryogenic character and embryo production. Cultures were initiated by culture of hypocotyl, cotyledon and root explants on MS (Murashige and Skoog 1962) medium supplemented with 1.0 mg·l−1 Kinetin and 0.5 mg·l−1 2,4-D, later transferred and maintained in liquid MS medium with 1.0 mg·l−1 Dicamba, 0.1 mg·l−1 NAA, 2.0 mg·l−1 BAP and 80.0 mg·l−1 AS. Regeneration medium included 0.0–1.0 mg·l−1 GA3+0.0−2.0 mg·l−1 Kin.+0.0−160 mg·l−1 AS.In these culture conditions, the effect of the explant was found to be the most important factor. The curve of growth, growth coefficient and % of participation of various size aggregates differed in the studied suspensions. Flow cytometry revealed various DNA content in nuclei from praembryogenic mass depending on the explant origin. To complete embryogenesis the medium was changed from liquid to solidified in the presence of the same plant growth regulators combination required. The most embryogenic culture appeared hypocotyl-derived and it yielded the highest number of somatic embryos. The suspension culture originating from root proliferated the highest number of embryogenic cell clusters but did not produce embryos for fraction 120–450 µm. One hundred mg of suspension of the fraction that was larger than 450 µm yielded 309, 175, 123 embryos for the following suspensions: root-, cotyledon-, hypocotyl-derived, respectively. Almost 50 % of non-deformed fully developed embryos from all studied suspensions passed conversion into germling stage and finally plants were regenerated.

Somatic embryogenesis of Gentiana genus IV.: Characterisation of Gentiana cruciata and Gentiana tibetica embryogenic cell suspensions

Experiments to characterise long-term embryogenic suspension cultures of Gentiana cruciata (L.) and G. tibetica (King) are reported. Cell suspensions of both species differed in the percentage of five selected fractions of cell aggregates, as well as in fresh and dry mass during three years of culture. In G. tibetica the ratio of cells in phase G2 to G1 was higher than in G. cruciata. The response of suspension cultures to GA3 (at 0, 1.49 or 2.89 µmol), kinetin (at 0, 2.32, 4.64 or 9.28 µmol) and adenine sulphate (at 0 or 434 µmol) was studied. The increase of kinetin concentration stimulated embryo production in suspensions of G. tibetica. Somatic embryo production in G. tibetica was significantly higher than in G. cruciata. In G. tibetica, the aggregate fraction >450 µm was at least four times more productive than the same fraction in G. cruciata suspensions.

Exploration of Cryo-methods to Preserve Tree and Herbaceous Fern Gametophytes

Fern gametophytes, derived from in vitro cultures, are a useful source of germplasm for ex situ conservation of endangered populations, scientific research using genetic stocks, mass production of ferns for plantings, and reproduction of species or hybrids that produce short-lived or infertile spores. Cryopreservation of gametophytic tissue is fairly established for mosses and liverworts, but is rare for ferns. The aim of this study was to develop methods for cryostorage of gametophytes from seven species of tree ferns originating from either the tropics or areas of mild winters and to compare these species’ amenability to cryopreservation with two herbaceous species that originate in areas where winters are harsher. Efficacy of three cryoprotection methods – vitrification, encapsulation/vitrification, and encapsulation/dehydration – was compared in terms of overall survival, time to sexual maturity (recovery rate), and preparation time to acquire tissues tolerant of cryoexposure. The standard vitrification procedure using PVS2 and PVS3 was ineffective and damaged the naked fern prothalia even without cryoexposure. Encapsulated in alginate beads prothalia during chemical desiccation with vitrification solutions allowing about 50% survival of gametophytes. The encapsulation/dehydration technique consistently gave high survival and rapid recovery in cryoexposed gametophytes. Two-week-long agar preculture with 0.25 M sucrose and 10 μM ABA enhanced survival or recovery rate and greater than 80% survival was achieved for eight of the nine species used in the study. Cryopreservability of fern gametophytes may be related to adaptions for longevity and desiccation tolerance in their natural habitats and totipotency of cells in the meristem.

Protein extraction from Ca-alginate encapsulated plant material for comparative proteomic analysis.

The extensive use of encapsulation material in biotechnology drove the need to develop analytical techniques for this type of material. This study focuses on the specific problems of protein extraction from Ca-alginate encapsulated plant material. Proteomics is one of the fast-developing analysis categories, specifically for stress resistance and developmental changes in plant material. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is essential for good results. The aim was to avoid preliminary manipulations and get good quality material for comparative proteome analysis technique 2DE. The phenol extraction method and the complex method with preliminary TCA precipitation, SDS buffer and phenol phase were compared with respect to the efficiency and quality of the resulting 2DE gel. The most appropriate method turned out to be the TCA/phenol method with the phenol fractioning technique adapted to the gentian cell suspension. It resulted in a high protein concentration and good quality sample that could be analyzed using the standard separation procedures of 2DE and spectrometric identification with high efficiency. The work presented here confirms the possibility of obtaining a sufficient protein sample for effective proteomic analysis from a small number of capsules.

Systems of Plant Regeneration in Gentian In Vitro Cultures

This chapter reviews the development of plant tissue culture and biotechnology of gentians during the last thirty years. The majority of 30 species studied belong to the genus Gentiana and those gentians are included into European flora. Biochemical studies aimed secondary metabolites production are not presented in the chapter. Explants from seedling were most frequently used for culture initiation. Differences between particular organs of a few-day-old seedling are significantly different in the presence of MS medium. Leaves from in vitro culture plants were used to describe their morphogenic potential and as a source of the protoplasts for somatic hybridization and transformation. The culture of floral explants helps to get interspecies hybrids and haploids with improving floriculture breeding programs. The shoot and root organogenesis and shoot multiplication play key role in the vegetative propagation of gentians. Embryogenic cultures on semi-solid and in liquid helped to undertake many subjects concerning somatic embryogenesis per se and exploration of embryogenic cell suspension for somatic cell genetic manipulation.

Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum × grandiflorum /Ramat./Kitam.

Cryopreservation is widely applied to many economically important species excluding chimera plants which are problematic for long-term conservation. Their storage problems can be circumvented only by cryopreserving meristems. This study looked at the morphogenetic response of shoot tips of periclinal chimera chrysanthemum ‘Lady Orange’ and ‘Lady Salmon’, as well as the solid mutant ‘Richmond’, that were cryopreserved by encapsulation-dehydration technique. By applying 10xa0µM ABA in the preculture medium followed by 4-day-long dehydration treatment, the explant survival reached up to 67%. Besides the stimulation of typical single shoot recovery, cryopreservation led to direct or indirect multiple shoot formation, shoot malformation, as well as inhibited their spontaneous rooting. Microscopic analysis revealed three types of structural damages of shoot tips which can correspond with their morphogenetic response in recovery culture. No influence of cryostorage on the acclimatisation efficiency of the recovered chrysanthemums was observed.