In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

In vitro cultivation of donor quince shoots affects subsequent morphogenesis in leaf explants

The effect of in vitro cultivation of donor shoots on subsequent morphogenesis in leaf explants of quince (Cydonia oblonga Mill.) clone BA29 was investigated. Proliferating donor shoots were cultured in ventilated or closed vessels under different photosynthetic photon flux densities (PPFD; 200 and 100 μmol m⁻² s⁻¹) with 0, 15, 30 g dm⁻³ sucrose. Shoots grown in ventilated vessels, especially with sucrose at 15 or 30 g dm⁻³, were better developed with fully expanded leaves compared to those in standard closed vessels. Leaves collected from pre-treated donor shoots were used to assess regeneration capacity. Somatic embryo production was highest in leaves harvested from shoots cultured in closed vessels with 30 g dm⁻³ sucrose and in ventilated vessels with 15 and 30 g dm⁻³ sucrose and under high PPFD which was, in comparison with the control treatment (closed vessel, 30 g dm⁻³ sucrose and low PPFD), about 2 to 2.5 times higher. A similar response was observed for root regeneration.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA), or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.     

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.     

Increased cold tolerance in Arabidopsis thaliana transformed with Choristoneura fumiferana glutathione S-transferase gene

A glutathione S-transferase (GST) gene cloned from the lepidopteran spruce budworm, Choristoneura fumiferana Clem. was transformed into the model plant Arabidopsis thaliana. The CfGST-transgenic and wild-type A. thaliana were subjected to 4 and 10 °C for 48 h and their cold resistance was studied. The GST activity of the transgenic plants was 46.6 and 35.7 % higher than that of the wild-type plants after 48 h under 4 and 10 °C, respectively. Relative membrane permeability and malondialdehyde content in the transgenic plants were lower while contents of the chlorophyll and proline were higher than those in the wild-type plants under 4 and 10 °C. The survival rate of the transgenic plants was 43.7 % for 24 h under 0 °C, while survival rate of wild-type plants was 28.3 %. The results indicated that the insect GST could enhance cold resistance in the transgenic A. thaliana.     

Cadmium-induced oxidative damage and antioxidant responses in Brassica juncea plants

Indian mustard (Brassica juncea L. cv. Vitasso) plants exposed to 10, 30, 50 and 100 μM of Cd for 5 d in hydroponic culture were analysed with reference to the distribution of Cd²⁺, the accumulation of biomass and antioxidants and antioxidative enzymes in leaves. Cd induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. Cd induced a decrease in catalase (CAT) and guiacol peroxidase (GPX) activities but an increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities. Enhancement in dehydroascorbate reductase (DHAR) activity was also at 10 μM Cd. Glutathione reductase (GR) activity showed pronounced stimulation after all treatments, but glutathione S-transferase (GST) and glutathione peroxidase (GPOX) activities decreased. The effectiveness of ascorbate-glutathione cycle (AGC) was determined by the ratio of ascorbate to H₂O₂. This ratio decreased in the Cd-treated leaves which indicated that the cycle was disordered.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.     

Different levels of inbreeding depression between outcrossing and selfing Serapias species

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.     

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.     

Nitrate reduction in ramets of a clonal plant Eichhornia crassipes responding to nitrate availability during clonal growth stage

Effect of nitrate availability on nitrate reduction was examined in inter-connected ramets of invasive clonal plant Eichhornia crassipes grown with two nitrate supply regimes during different clonal growth stage. Increase of nitrate availability accelerated nitrate reductase activity (NRA) in parent and offspring ramets of E. crassipes, and there was greatly different pattern in inter-connected ramets during clonal growth stage. Leaf NRA was lower in offspring than that in parent ramets in phase 1, while significantly higher leaf NRA in offspring ramets was detected during phase 2. The results indicated NRA in inter-connected ramets of E. crassipes was highly dependent on nitrate availability and growth stage.     

Polyamine metabolism in hants : Arginine and omithine decarboxylase activity in ripeningTrlticum durum seeds

The pattern of the activity of arginine decarboxylase (ADC) and omithine decarboxylase (ODC) involved in polyamine synthesis in ripening wheat seeds was examined. The aim was to study the polyamines and the activity of the two enzymes in correlation with the growth processes occurring in the developing wheat seeds. The results obtained showed a very different pattern of polyamine content in the two organs of caryopsis, and that the two enzymes in the embryos have a higher activity than in the endosperms. Moreover, while in the embryos the ADC exhibits higher activity than the ODC, in the endosperms the activity of ODC is about similar to that of ADC. This pattern is discussed in relation to the different histological characteristics of embryo and endosperm tissues during seed development.     

RAPD markers associated with quercetin accumulation in Psidium guajava

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.     

Diurnal changes in chlorophyll fluorescence and light utilization in Colocasia esculenta leaves grown in marshy waterlogged area

Diurnal cycle of chlorophyll fluorescence parameters was done in Colocasia esculenta L. (swamp taro) grown in marshy land under sun or under shade. The sun leaves maintained higher electron transport rate (ETR) and steady state to initial fluorescence ratio (F_s/F₀) than shade leaves. In spite of lower ETR, higher photochemical quenching (PQ), and effective quantum yield of photosystem 2 (Φ_PS2) was evident in shade plants compared to plants exposed to higher irradiance. ETR increased linearly with increase in irradiance more under low irradiance (r ² = 0.84) compared to higher irradiance (r ² = 0.62). The maximum quantum yield of PS 2 (F_v/F_m) did not differ much in sun and shade leaves with the exception of midday when excess of light energy absorbed by plants under sun was thermally dissipated. Hence swamp taro plants adopted different strategies to utilize radiation under different irradiances. At higher irradiance, there was faster decline in proportion of open PS 2 centers (PQ) and excess light energy was dissipated through non-photochemical quenching (NPQ). Under shade, absorbed energy was effectively utilized resulting in higher Φ_PS2.     

Modelling the dynamics of the electron transport rate measured by PAM fluorimetry during Rapid Light Curve experiments

We propose a dynamic model specifically designed to simulate changes in the photosynthetic electron transport rate, which is calculated from fluorescence measurements when plants are exposed, for a short time, to a series of increasing photon flux densities. This model simulates the dynamics of the effective yield of photochemical energy conversion from the maximum and natural chlorophyll fluorescence yields, taking into account a cumulative effect of successive irradiations on photosystems. To estimate a characteristic time of this effect on photosystems, two series of experiments were performed on two benthic diatom culture concentrations. For each concentration, two different series of irradiations were applied. Simplified formulations of the model were established based on the observed fluorescence curves. The simplified versions of the model streamlined the parameters estimation procedure. For the most simplified version of the model (only 4 parameters) the order of magnitude of the characteristic time of the residual effect of irradiation was about 38 s (within a confidence interval between 20 and 252 s). The model and an appropriate calibration procedure may be used to assess the physiological condition of plants experiencing short time-scale irradiance changes in experimental or field conditions.     

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.     

MPEC: an important gene in the chlorophyll biosynthesis pathway in photosynthetic organisms

One of the least understood enzymatic steps in chlorophyll biosynthesis is the formation of isocyclic ring, which is catalyzed by the Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase that is involved in the conversion of MgPME to protochlorophyllide. Several genes encoding part of this enzyme have been identified and functional analysis of them has been performed. The enzyme plays important roles in higher plants and photosynthetic bacteria. The review focuses on the current knowledge of MgPME cyclase coding genes, with emphasis on their organization, expression pattern, and functional analysis obtained from mutants.     

Flooding tolerance and genetic diversity in populations of <Emphasis Type="Italic">Luehea divaricata</Emphasis>

We investigated some aspects of flooding tolerance in two riparian populations (exposed and no exposed to flooding) of Luehea divaricata C. Martius. Plants derived from seeds collected in each population were submitted to flooding (30 and 60 d), submergence and re-aeration treatments. Plants exposed to flooding showed development of aerenchyma, hypertrophic lenticels and new adventitious roots. Interestingly, whereas the plants originated from population naturally exposed to flooding developed some of these alterations more markedly, they could not survive when totally submerged. The random amplified polymorphic DNA (RAPD) markers, showed a significant difference between populations, suggesting that seasonal flooding on riparian populations of L. divaricata has been selecting individuals who are more adapted to survive in these conditions.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.