We used simple sequence repeat markers and 25 morphological characters to characterize 18 Tunisian fig (Ficus carica L.) cultivars. Morphological traits suggested a high level of variation in the germplasm. Principal component analysis (PCA) differentiated the studied cultivars. In the derived dendrogram the cultivars clustered independently of their geographical origin and sex of trees. Simple sequence repeat (SSR) markers were used to compare genetic polymorphism with the observed phenotypic variation. Using six microsatellite primers, 39 alleles and 59 genotypes were identified. The high values of polymorphism information content (PIC), ranging from 0.67 to 0.85, confirmed the effectiveness of microsatellite analysis for determining molecular polymorphism and characterizing the studied cultivars. Multilocus genotyping unambiguously distinguished all the cultivars. The ability of each type of feature to differentiate cultivars of this crop is discussed.
Growth and photosynthetic characteristics, inducibility of the CAM pathway and the functioning of the antioxidant defense system were investigated in Rosularia elymaitica (Crassulaceae) under drought and UV stresses. Drought did not substantially affect the growth of the plants, but it significantly reduced leaf thickness as well as osmotic potential, water potential and relative water content. In contrast, UV radiation treatment affected neither growth nor the water relations of leaves. Water limitation for 12 days caused a significant increase in nighttime PEPC and NAD-MDH activity and an increase in Δtitratable acidity relative to well-watered plants. The nighttime CO2 net assimilation rate increased significantly in drought-stressed plants but was still negative, resembling a C3-like pattern of gas exchange. Twenty days of UV treatment, increased Δtitratable acidity slightly and increased only daytime PEPC activity, and did not affect other parameters of carbon metabolism. As judged by maintenance of membrane integrity and stable amounts of H2O2 under UV stress, the antioxidant defense system effectively protected the plants against UV radiation. In contrast, oxidative stress occurred under severe drought stress (20 days of withholding water). Except for higher daytime APX activity in the UV-treated plants, enzyme activity in the control and in the drought- and UV-stressed plants did not show any diurnal fluctuation during 24 h. Temporal changes in Δtitratable acidity and ΔPEPC activity coincided closely with those of antioxidant enzymes; both started to increase after 12 days of drought stress. These results indicate that drought stress but not UV radiation induced the CAM-cycling pathway in R. elymaitica.
The full-length cDNA of LeTIR1 gene was isolated from tomato with EST-based in silico cloning followed by RACE amplification. LeTIR1 contained an open reading frame (ORF) 1872 bp long, encoding 624 amino acid residues. The predicted protein LeTIR1 had one F-box motif and eleven leucine-rich repeats (LRRs), all of which are highly conserved in TIR1 proteins of other plant species. Phylogenetic analysis showed that the LeTIR1 protein shared high similarity with other known TIR1 proteins. Both sequence and phylogenetic analysis suggested that LeTIR1 is a TIR1 homologue and encodes an F-box protein in tomato. Semi-quantitative RT-PCR indicated that LeTIR1 was expressed constitutively in all organs tested, with higher expression in stem than root, leaf, flower and fruit. Its expression level was positively correlated with the auxin distribution in stem or axillary shoot, and was induced by spraying exogenous IAA.
We investigated the germination requirements of Androsace villosa L. (Hairy Androsace), which spreads on limestone or granite screes or ledges of rocky or turfy slopes and hilltops of the alpine zone. With seeds collected from Uludag Mt. (Bursa, Turkey, 2200-2300 m a.s.l.), germination was studied in fresh seeds, seeds subjected to short-time moist chilling (15 d, +4°C), to GA3 (100, 150 and 250 ppm), and to chilling plus GA3. The hormone and moist chilling treatments were carried out in continuous darkness (20°C) and under a 12 h photoperiod at 20/10°C. Seeds maintained in darkness gave higher germination percentages than seeds maintained under a photoperiod. Germination rates rose to 90-97% with 100-250 ppm GA3 and short-time moist chilling in continuous darkness (20°C). Seeds germinated rapidly under a combination of GA3 and short-time moist chilling in continuous darkness, generally giving the lowest mean germination times (4.4-5.0 d) among the treatments.
Our survey of data collected in the Chromosome Number Database for Polish angiosperms indicated that the 1,498 species with chromosome counts represent 40% of the total angiosperms (3,719) occurring in Poland, including 1,205 native species (53% of native species) and 194 anthropophytes (56% of anthropophytes). The chromosome numbers are known for all native species occurring in Poland within 298 genera and 46 families, and for all anthropophytes from 79 genera and 11 families. The remaining angiosperm groups are less explored: chromosome counts from Poland are known for 9% of cultivated species and 5% of ephemerophytes. According to generic basic chromosome numbers, 46.44% of Polish angiosperms have been classified as polyploid. By three different threshold methods, the contribution of polyploid plants to the Polish flora is 64.64%, 50.89% or 42.89%. Polyploidy is more common among indigenous than non-indigenous plants, and the ploidy distribution among plants from the Polish Tatras does not differ significantly from that observed in the rest of native Polish plants.
Members of the Nyctaginaceae Jussieu. are distributed throughout southern Africa. Eight species of the family occur naturally in arid parts of Namibia. These species have acquired the ability to survive and reproduce in these dry conditions. They are xerophytes, which have been described as drought evaders, avoiders, and drought-tolerant. In the Nyctaginaceae, Boerhavia deserticola, B. hereroensis, Commicarpus helenae and C. squarrosus are thought to be drought avoiders. In this study we investigated their stem, leaf and anthocarp anatomy for adaptations to arid environments. The results indicated that the four species are avoiders, with modifications of the trichomes, secretions, crystals, secondary growth, Kranz mesophyll, water storage cells, tannins, mucilage, inner and outer stomatal ledges, large-diameter xylem vessels, and the presence of sclerenchyma in their stems, leaves and anthocarps. These adaptations enable the plants to tolerate arid conditions, conserve water and maintain a high photosynthetic rate, and aid seed dispersal.
To understand the molecular mechanism controlling in vitro plant morphogenesis, a culture system enabling induction of alternative morphogenic pathways (somatic embryogenesis, SE; shoot organogenesis, ORG) in a well defined population of somatic cells is needed. Arabidopsis is the most useful model plant for genomic studies, but a system in which SE or ORG can be induced alternatively in the same type of explant has not been proposed. Immature zygotic embryos (IZEs) of Arabidopsis provide the only explants with embryogenic potential, and have been recommended for studying mechanisms of SE induced in vitro. This study was aimed at defining culture conditions promoting induction of alternative morphogenic pathways: shoot ORG in IZE explants. The established protocol involves pretreatment of IZE explants with liquid auxin-rich callus induction (CIM) medium, followed by subculture on solid cytokinin-rich shoot induction medium (SIM). The method enables efficient shoot induction in Columbia (Col-0) and Wassilewskija (Ws), genotypes commonly used in molecular studies. During 3 weeks of culture up to 90% of Col-0 and 70% of Ws explants regenerated shoots via an indirect morphogenic pathway. We analyzed the qRT-PCR expression patterns of the LEC (LEC1, LEC2 and FUS3) genes, the key regulators of Arabidopsis embryogenesis, in the IZE explants induced to promote shoot ORG. The sharp decline of LEC expression on SIM medium confirmed that culture of Arabidopsis IZE explants enables experimental manipulation of the morphogenic response of somatic cells. A scheme illustrating various in vitro morphogenic responses of IZEs in relation to hormonal treatment is presented.
This paper is the first published report describing micropropagation of Carlina onopordifolia, using shoot tip and hypocotyl explants. The explants were excised from 10-day-old seedlings and transferred to proliferation medium supplemented with 6-benzylaminopurine (BA; 1.0 or 3.0 mg l-1), kinetin (Kn; 1.0 or 3.0 mg l-1) or zeatin (ZEA; 1.0 or 3.0 mg l-1) in combination with naphthaleneacetic acid (NAA; 0.1 mg l-1). The shoot tips were significantly better than hypocotyls as initial material for shoot regeneration. For shoot multiplication, MS medium supplemented with BA proved superior to the other cytokinins tested. Medium supplemented with 1.0 mg l-1 BA gave the highest shoot propagation frequency (66.9%) and number of shoots per explant (2.5). Single shoots were separated from each other and rooted on MS supplemented with IBA for the whole period of culture, with longor short-pulse IBA application. The highest rooting frequency (84.8%) and root number (18.8) were for shortpulse (1 min) 1000 mg l-1 IBA solution. The higher IBA concentration stimulated callus formation and the development of short roots. The shoots were transferred to MS medium without growth regulators. Survival was highest (54.4%) for the plants from the short-pulse 100 mg l-1 IBA treatment. After 8 weeks of acclimatization the plantlets were removed to field conditions and grew normally.
This study examined the effects of UV-B radiation and allelochemical stress induced by ferulic acid (FA) on the activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) at metabolic and molecular levels in two cucumber genotypes differing in tolerance to cold and disease, in order to determine any interaction between stress effects and genotype response. Stresses were applied simultaneously, sequentially, and singly. In both genotypes, several days of UV radiation retarded growth up to 36%. The effect of FA was not significant. The response to a particular stress, including the effect on PAL activation, was enhanced by simultaneous application of the two stresses. PAL transcription was not correlated with the increase of PAL activity. Exposure to UV-B, FA, and combined UV-B and FA was detrimental to both genotypes but to different extents. The response was not correlated with the genotype of cold and disease sensitivity. PAL activity and its transcription seem to be involved in UV and allelochemical stress, but not related to the plants' tolerance of these stresses.
Centaurium erythraea plants obtained by indirect organogenesis are described in the paper. The plants were initiated from a single adventitious shoot regenerated from callus derived from the cotyledon of a 30-day-old seedling. The shoot was multiplied on MS medium supplemented with IAA (0.1 mg·L-1) and BAP (1.0 mg·L-1). The multiplication rate (28 shoots per culture within 4 weeks) was highest at the first subculture and decreased in further subcultures. The shoots were rooted on MS medium. The effect of IBA (0.1 mg·L-1) on the number of shoots forming roots differed depending on the composition of the basal medium (MS). The rooted shoots were transplanted to soil and grown in a greenhouse with 90% effectiveness. RAPD analysis was done with adventitious shoots of C. erythraea from in vitro culture. In shoots and whole plants regenerated from the callus tissue, secoiridoid content was determined by the HPLC method. We showed significant differences in morphology (leaf size, fresh and dry weight and height of plants) and changes in the DNA profiles as compared to earlier reports for shoot tip-derived shoots and plants of C. erythraea, but the two groups of plants biosynthesized the same qualitative pattern and similar levels of secoiridoids, up to 150 mg·g-1 dry weight; the increased biomass of plants regenerated from callus tissue makes them a better source of secondary metabolites.
Roots of Codonopsis pilosula (Franch.) Nannf. are among the most popular Chinese herbal medicines, exhibiting various beneficial activities which support immunity and stress resistance. The plant shows high intraspecific genetic variation. There is a need for effective vegetative propagation methods yielding high and sustainable quality. Here we report a micropropagation method using axillary shoot proliferation. Nodal segments from aseptically germinated plants were inoculated on modified MS media enriched with different concentrations of cytokinins: benzyladenine, kinetin (1, 4, 10 or 20 μM) or thidiazuron (1, 4 or 8 μM), with or without the auxin NAA (1 μM). Axillary bud break was initiated most efficiently on media with 1 or 4 μM BA and 1μM NAA. Shoot number increased markedly in subsequent cycles of harvesting and transfer to fresh 1 μM BA and NAA medium, leading to the maximum 69 shoots (mean 38.16±4.35) from a single nodal explant in the fourth harvest. The shoots were successfully (>98% efficiency) rooted in MS medium containing high sucrose (60 g/L) and 5 μM IAA, and acclimatized to soil cultivation with a survival rate of 90%. These results can be used to establish a simple and commercially viable protocol for mass propagation of C. pilosula for plantations or breeding.
In plants belonging to the Ranunculaceae the floral nectaries may differ in origin, location in the flower, shape and structure. In many cases they are defined as modified tepals or modified stamens. The nectary organs in this family are frequently termed "honey leaves," and staminodial origin is attributed to them. Gynopleural and receptacular nectaries are rarely found in Ranunculaceae. To date there are no reports on the structure of the nectary organs in plants of the genus Pulsatilla. We used light and scanning electron microscopy to study the location and structure of the nectaries in Pulsatilla slavica and P. vulgaris flowers. The staminodial nectaries were found to be nectar-secreting organs. The number of stamens per flower (102-398) increases with plant age. The share of staminodes is 12-15%. The staminodes are composed of a filament and a modified head. They are green due to the presence of chloroplasts in the epidermal and parenchymal cells. The parenchymal cells are in a loose arrangement. Stomata (3-20), through which nectar exudation occurred, were found only in the abaxial epidermis of the staminode head. The stomata are evenly distributed and have well-developed outer cuticular ledges. Some of them are immature during nectar secretion, with their pores covered by a layer of cuticle. During the activity of the nectariferous organs in the flowers, primary (on the staminode surface) and secondary nectar (at the base of tepals) are presented. The staminodes of the two Pulsatilla species show similar structural features and have similar shares in the androecium.
Megasporogenesis and female gametophyte development were investigated in ovules of the everbearing strawberry Fragaria x ananassa Duch. cv. Selva. Observations of thin sections revealed that ovule development starts from the formation of a nucellus and coincides in time with the beginning of receptacle overgrowth. The most characteristic feature during nucellus differentiation is the formation of a multicellular archesporium, beginning from at least two cells. Analysis of female gametophyte development indicated that in addition to the meiotic mode, female gametophytes develop by an apomeiotic mode of Antennaria type. Asynchronous development of female gametophytes of different origin occurs. The mature, eight-nucleate, seven-celled female gametophyte of meiotic origin is cylindrical and slightly curved. It occupies the central part of the nucellus. The egg apparatus, consisting of an egg cell and two synergids, is formed in the micropylar part of the female gametophyte; the opposite chalazal pole is occupied by antipodal cells. Besides the ovule in which only one seven-celled female gametophyte finally develops, ovules with a different number of cells were observed to initiate female gametophyte development. Some ovules contain a nucellus with a tetrad of linearly arranged megaspores surrounded by enlarged cells, each of which has the potential to develop into an apomictic female gametophyte. After degeneration of some post-meiotic cells or developmentally advanced female gametophytes, some of the chalazal cells initiated female gametophyte development.
The arbuscules of mycorrhizae develop within apoplastic compartments of the host plant, as they are separated from the cell protoplast by an interfacial matrix continuous with the plant cell wall. Expansins are proteins that allow cell wall loosening and extension. Using fluorescence and electron microscopy we located the NtEXPA5 epitopes recognized by polyclonal antibody anti-NtEXPA5 in mycorrhizal tobacco roots. The expansin protein was localized mainly within the interfacial matrix of intracellular hyphae, arbuscule trunk and main branches. NtEXPA5 proteins were detected neither within the interface of collapsing arbuscule branches nor in non-colonized cortex cells. In plant cell walls, expansin protein was detected only at the penetration point and in the parts of cell walls that adhered firmly to fungal hyphae growing intracellularly. For the first time, NtEXPA5 protein was localized ultrastructurally in hyphae growing intracellularly at the interface of the hypha tip and sites of bending. The novel localization of NtEXPA5 protein suggests that this protein may be involved in the process of arbuscule formation: that is, in promoting apical hyphal growth and arbuscule ramification, as well as in controlling the dynamic of arbuscule mycorrhiza development.
The authors report the first discovery of diploid populations of Hieracium naegelianum Panč. subsp. naegelianum and H. naegelianum subsp. ljubotenicum Behr & Zahn., and give the first chromosome counts for H. cernuum Friv., H. gymnocephalum Griseb. ex Pant., H. sparsum Friv., Pilosella pavichii (Heuff.) Holub and P. serbica (F. W. Schultz & Schultz-Bip.) Szeląg from Macedonia and/or Montenegro. A diploid chromosome count for Hieracium renatae Szeląg is confirmed based on material from the whole distribution range of the species. An emasculation experiment showed that all the analyzed diploid Hieracium taxa reproduce sexually.
Steroidal saponins isolated from many plant species belonging to Monocotyledones display potent cytotoxic activity towards many human tumor cells. We examined the cytotoxic effects of crude Paris quadrifolia extract for the first time, testing isolated saponin-rich fractions against four different human cell lines using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxic activity was tested against human promyelocytic leukemia (HL-60) cells, human cervical adenocarcinoma (HeLa) cells and human breast cancer (MDA-MB-468) cells. Human skin fibroblasts were used as non-neoplastic control cells. Our results show significant activity of the weakly water-soluble solid residue and butanolic fraction against HL-60 and HeLa cells. The solid residue exerted cytotoxicity against all tested cell lines.
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