Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

Assessment of genetic stability of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Dictyospermum ovalifolium</Emphasis>

In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Genetic relationships among <Emphasis Type="Italic">Hystrix patula,H. duthiei</Emphasis> and <Emphasis Type="Italic">H. longearistata</Emphasis> according to meiotic studies and genome-specific RAPD assay

Hybrids including Hystrix patula, H. duthiei and H. longearistata were obtained and genetic relationships among them were studied. Meiotic pairing in hybrids of H. duthiei × Psathyrostachys juncea (Ns), H. longearistata × Psa. juncea (Ns), Leymus multicaulis (NsXm) × H. duthiei, L. multicaulis (NsXm) × H. longearistata, Elymus sibiricus (StH) × H. patula, Roegneria ciliaris (StY) × H. patula, R. ciliaris (StY) × H. duthiei and R. ciliaris (StY) × H. longearistata averaged 5.76, 5.44, 11.94, 10.88, 10.08, 3.57, 0.46 and 0.90 bivalents per cell, respectively. The results indicated that H. duthiei and H. longearistata had the NsXm genomes of Leymus, while H. patula contained the StH genomes and had a low genome affinity with the StY genomes of Roegneria. Results of genome-specific RAPD assay were comparable with the chromosome pairing data. According to the genomic system of classification in Triticeae, H. patula should be considered as Elymus hystrix L., while H. duthiei and H. longearistata as Leymus duthiei and Leymus duthiei ssp. longearistata, respectively.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Effect of growth regulators and ethylmethane sulphonate on growth,and chlorophyll,sugar and proline contents in <Emphasis Type="Italic">Dracaena sanderiana</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m⁻³). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET₁ and ET₂) comparatively to control and other (ET₃, ET₄ and ET₅) lines. Calli of ET₁ line showed high regeneration potential on MS medium with N ⁶-benzylaminopurine (BAP; 1.75 g m⁻³). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA₃; 0.4 g m⁻³). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m⁻³).     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> assays for osteoclast apoptosis

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.     

Interspecific hybridization of <Emphasis Type="Italic">Cucumis anguria</Emphasis> and <Emphasis Type="Italic">C. zeyheri via</Emphasis> embryo-rescue

Embryo-rescue was used to facilitate interspecific hybridization of Cucumis anguria L. and C. zeyheri Sond. Embryos were excised from developing fruits at one week intervals for six weeks after hand pollination. Medium containing coconut water was the most suitable for initial germination, and a medium with ascorbic acid was the best for embryo development and plant recovery. Viable plants were obtained from embryos and these plants showed morphological characteristics different from both parents. The analysis of the leucine aminopeptidase (LAP) locus revealed three hybrid types, H1.1, H1.2 and H2.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Ginsenoside production,growth and cytogenetic characteristics of sustained <Emphasis Type="Italic">Panax japonicus</Emphasis> var. <Emphasis Type="Italic">Repens</Emphasis> cell suspension culture

Cytophysiological and cytogenetic characteristics of cell suspension culture of Panax japonicus var. repens were studied in relation to the accumulation of ginsenosides (GSs). The minimal time of cell number doubling was 1.3 ± 0.1 d and cell number increased 7 to 8-fold during growth cycle. The cell culture can be considered as aneuploid with about tetraploid (46–60 chromosomes) modal class. Upon long-term cultivation, the total content of GSs considerably increased and maximal concentration of GSs was 2.2 %(d.m.). The ratio of seven major GSs only slightly altered both over each and different subcultures. The overall amount of GSs of Rg-group significantly exceeded that of Rb-group. Cell volume and the number of large cellular aggregates with the higher proportion (by 20 %) of parenchymal cells increased late in the subculture. In this time the population contained about 20 % of the cells with doubled amount of nuclear DNA and accompanied with elevation in the GS content. These data prompted us to suggest that biosynthesis of GSs has a link with cell differentiation. In memory of Prof. R.G. Butenko     

Ca<Superscript>2+</Superscript> reduces the effect of hypoxia in mosses <Emphasis Type="Italic">Mnium undulatum</Emphasis> and <Emphasis Type="Italic">Polytrichum commune</Emphasis>

Gametophores of mosses Mnium undulatum and Polytrichum commune were submerged in distilled water or in calcium chloride solution (0.9 mM Ca²⁺) to induce hypoxia. The net photosynthetic (P_N) and dark respiration rate (R_D) were measured in the air containing 300–400 μmol(CO₂)·mol⁻¹(air) and 0.21 mol(O₂)·mol⁻¹(air). P_N of M. undulatum gametophores decreased to 58 % of the control after 1-h submersion in water, whereas to 80 % of the control in P. commune gametophores. A smaller decrease in P_N was observed when the gametophores were immersed in CaCl₂ solution. In hypoxia, R_D in the tested mosses species was a little higher than in the control.     

Photosynthetic characteristics of an endangered species <Emphasis Type="Italic">Camellia nitidissima</Emphasis> and its widespread congener <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Saturation (SI) and compensation (CI) irradiances [μmol(photon) m⁻² s⁻¹] were 383.00±18.40 and 12.95±0.42 for wild C. nitidissima (in mid-July) and 691.00±47.39 and 21.91±1.28 for wild C. sinensis, respectively. C. nitidissima is a shade tolerant species, whereas C. sinensis has a wide ecological range of adaptability to irradiance. Both wild and cultivated C. nitidissima demonstrated low maximum net photosynthetic rate, maximum carboxylation rate, maximum electron transfer rate, and SI, which indicated low photosynthesis ability of leaves that were unable to adapt to strong irradiance environment. Both C. nitidissima and C. sinensis demonstrated strong photosynthetic adaptabilty in new environments. Hence proper shading may raise photosynthetic efficiency of cultivated C. nitidissima and promote its growth.     

A new tip homolog, <Emphasis Type="Italic">ShTIP</Emphasis>, from <Emphasis Type="Italic">Salicornia</Emphasis> shows a different involvement in salt stress compared to that of TIP from <Emphasis Type="Italic">Arabidopsis</Emphasis>

To obtain an insight into the comprehensive molecular characteristics of the salt tolerance mechanism, we performed a screening for salt inducible genes in a halophytic plant, Salicornia herbacea, using mRNA differential display. A comparative analysis of gene expression in Salicornia grown in control and salt-stressed conditions led to the detection of a gene that was induced by salt. Both sequence analysis and a subsequent database search revealed that this gene was highly homologous to tonoplast intrinsic proteins (TIPs) from a variety of plant species. This gene, designated as ShTIP, is 1014 bp in size and contains a coding region of 762 nucleotides, which encodes a protein of 254 amino acids. Northern blot analysis revealed that ShTIP was predominantly expressed in shoots under normal conditions. However, salt stress induced high expression of ShTIP in both the shoots and roots. The expression of ShTIP in a salt-sensitive calcineurin-deficient yeast mutant (cnbΔ) resulted in a resistance to the high salt conditions. In addition, we compared the expression of a TIP gene in Arabidopsis with that of ShTIP under different conditions and found that the Salicornia TIP has a different regulatory mechanism for adapting to salt stress conditions compared with the glycophyte Arabidopsis TIP. These results indicate that ShTIP plays an important role in salt tolerance.     

Salt stress effects on growth,pigments, proteins and lipid peroxidation in <Emphasis Type="Italic">Salicornia persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>

The effects of NaCl stress on growth, water status, contents of protein, proline, malondialdehyde (MDA), various sugars and photosynthetic pigments were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under NaCl (0, 100, 200, 300, 400, 500 and 600 mM) on Murashige and Skoog medium for 45 d. The shoot growth of both species increased under low NaCl concentration (100 mM) and then decreased with increasing NaCl concentrations. In contrast to S. persica, root length in S. europaea reduced steadily with an increase in salinity. Proline content in S. persica was higher than in S. europaea at most NaCl concentrations. Proline, reducing saccharide, oligosaccharide and soluble saccharide contents increased under salinity in both species. In contrast, contents of proteins and polysaccharides reduced in both species under salt stress. MDA content remained close to control at moderate NaCl concentrations (100 and 200 mM) and increased at higher salinities. MDA content in S. europaea was significantly higher than S. persica at higher salinities. Salt treatments decreased K⁺ and P contents in seedlings of both species. Significant reduction in contents of chlorophylls and carotenoids due to NaCl stress was also observed in seedlings of both species. Some differences appeared between S. persica and S. europaea concerning proteins profile. On the basis of the data obtained, S. persica is more salt-tolerant than S. europaea.     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.