Response Of Explants Research Articles

Antimicrobial peptides and pro‐inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology.

In Vitro Micropropagation of the Ornamental Plant Dieffenbachia-A Review

Ornamental industry has applied immensely in vitro propagation approach for large-scale plant multiplication at very high rates of elite superior varieties. As a result, hundreds of plant tissue culture laboratories have come up worldwide. Dieffenbachia species are popular foliage potted plants used in interiorescapes of homes, offices, and malls throughout the world. Most of Dieffenbachia species are now propagated by tissue culture for better utilize of species and expedite plant improvement. This review paper summarizes valuable literature on in vitro techniques including type of explants used, media optimized, ways of propagation and improvement through 45 years of research on Dieffenbachia spp. Which were provide basis for future studies such as genetic transformation for breeding aims, develop new cultivars, develop disease-resistant plants and overcome the environmental obstacles. There is a need for more application of the plant tissue culture techniques on Dieffenbachia to investigate the responses of different cultivars and explants to variable culture media.

Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

Optimisation ofin vitroregeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants - ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined effects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l−1) dramatically decreased necrotic response of explants. However, citric acid, used together with ascorbic acid completely inhibited shoot growth in shoot apex cultures. Glutathion evoked hyperhydricity of explants. Activated charcoal induced rooting on media which are commonly used for shoot proliferation. Combination of acetosyringone with antioxidants influenced shoot proliferation, except of variant with ascorbic acid. Citric acid was the best and universal antioxidant in faba beanin vitrocultures and its use is recommended for faba bean genetic transformation experiments.

Characterization of Interferon Responses in Human Placental Explants by Whole Mount Immunofluorescence

Characterization of Interferon Responses in Human Placental Explants by Whole Mount Immunofluorescence

LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis

The LEAFY COTYLEDON2 (LEC2) transcription factor with a plant-specific B3 domain plays a central role in zygotic and somatic embryogenesis (SE). LEC2 overexpression induced in planta leads to spontaneous somatic embryo formation, but impairs the embryogenic response of explants cultured in vitro under auxin treatment. The auxin-related functions of LEC2 appear during SE induction, and the aim of the present study was to gain further insights into this phenomenon. To this end, the effect of LEC2 overexpression on the morphogenic responses of Arabidopsis explants cultured in vitro under different auxin treatments was evaluated. The expression profiles of the auxin biosynthesis genes were analysed in embryogenic cultures with respect to LEC2 activity. The results showed that LEC2 overexpression severely modifies the requirement of cultured explants for an exogenous auxin concentration at a level that is effective in SE induction and suggested an increase in the auxin content in 35S::LEC2-GR transgenic explants. The assumption of an LEC2 promoted increase in endogenous auxin in cultured explants was further supported by the expression profiling of the genes involved in auxin biosynthesis. The analysis indicated that YUCCAs and TAA1, working in the IPA-YUC auxin biosynthesis pathway, are associated with SE induction, and that the expression of three YUCCA genes (YUC1, YUC4 and YUC10) is associated with LEC2 activity. The results also suggest that the IAOx-mediated auxin biosynthesis pathway involving ATR1/MYB34 and CYP79B2 does not seem to be involved in SE induction. We conclude that de novo auxin production via the tryptophan-dependent IPA-YUC auxin biosynthesis pathway is implicated in SE induction, and that LEC2 plays a key role in this mechanism.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1892-2) contains supplementary material, which is available to authorized users.

Effects of exogenous l-arginine on in vitro rooting, chlorophyll, carbohydrate, and proline concentrations in the sweet cherry rootstock M × M 14 (Prunus avium L. × Prunus mahaleb L.)

The effects of indole-3-butyric acid (IBA) alone and in combination with l-arginine on the morphogenic and biochemical responses in shoot tip explants of the cherry rootstock M × M 14 (Prunus avium × Prunus mahaleb) were examined. The maximum root number per rooted explant (16), root fresh (FW) and dry (DW) weights, as well as the rooting percentage (100 %) were recorded when 2 mg l−1 IBA (alone) were applied. Including the lowest IBA concentration (0.5 mg l−1) with the lowest and highest l-arginine concentrations (0.5 and 2 mg l−1, respectively) resulted in the greatest root length. The maximum leaf chlorophyll concentration and shoot length of the initial explant were recorded when 0.5 mg l−1 IBA plus 2 mg l−1l-arginine were applied. In addition, l-arginine in combination with IBA (1 and 2 mg l−1) was found to suppress shoot FW and DW. On the other hand, l-arginine enhanced the promoting effect of IBA on both root length and leaf chlorophyll concentration. The carbohydrate and proline concentrations in leaves were not significantly altered with the application of IBA alone or in combination with l-arginine. On the other hand, the carbohydrate and proline concentrations in roots were decreased with the application of 1 and 2 mg l−1 IBA with l-arginine, resulting in the suppression of the promoting effects of IBA. It is clear from the findings that l-arginine has a direct effect on the in vitro rooting of M × M 14 explants, is involved in the function of the photosythetic apparatus, influences leaf chlorophyll content, participates in carbohydrate biosynthesis and metabolism, and is involved in proline accumulation both in leaves and roots.

Assessment of interferon responses in intact human placental explants using a novel whole mount immunofluorescence technique (P3205)

Abstract Pro-inflammatory cytokines required for immunity against non-self, such as interferons (IFNs), are expressed at the maternal-fetal interface during normal pregnancy. However, neither the placental cell targets nor the functions of these cytokines have been defined. We hypothesized that semi-allogeneic trophoblast cells (TBCs), which are the only cells derived from the fertilized egg that are in direct contact with maternal tissue, are hypo-responsive to IFNs in order to evade maternal immune recognition. Therefore, we developed a novel whole mount immunofluorescence technique to identify cellular targets of IFN-γ and IFN-α/β by examining expression of phosphorylated STAT-1 (pSTAT-1) in intact human placental explants. Using this technique we were able to identify the major cell populations present in the placental explants, including fetal macrophages and distinct subpopulations of TBCs. Neither the syncytiotrophoblast layer in direct contact with maternal blood, nor the underlying cytotrophoblast cells expressed pSTAT-1 in response to either IFN. In contrast, extravillous trophoblast cells, which are in direct contact with maternal decidual tissue, and fetal macrophages induced strong expression of pSTAT-1 in response to IFNs. The identification of differential IFN responsiveness in distinct human trophoblast cell subpopulations provides important new insights into the roles of IFNs in normal pregnancy and placental pathology and infection.

In vitro propagation and assessment of genetic stability of micropropagated Samanea saman (rain tree) using microsatellite markers

An efficient in vitro propagation of Samanea saman (rain tree) protocol has been successfully developed using nodal explants from a 20-year-old tree. Higher percentage (76 %) of explants produced up to five shoots per explant on Murashige and Skoog (MS) medium supplemented with 2 mg L−1 6-benzyladenine (BA), 0.1 mg L−1 gibberellic acid (GA3) and 100 mg L−1 casein hydrolysate after 3 weeks of culture. When explants were subcultured to fresh medium after harvesting first batch of shoots, more shoots could be generated (another eight shoots per explant). Shoot elongation was achieved (3 cm) when shoots were cultured on MS medium supplemented with 0.25 mg L−1 BA and 0.75 mg L−1 GA3. In vitro generated shoots rooted on MS medium fortified with 0.75 mg L−1 indole-3-butyric acid plus 0.1 % of activated charcoal. A higher percentage of explant response and shoots per explant were obtained on MS medium with BA and GA3. Each responsive nodal explant yields an average of 15 rooted plants within a period of 10 weeks. Rooted plantlets were successfully acclimatized in green house with a survival rate of 90 %. Micropropagated plants were tested for genetic stability using simple sequence repeats (SSR) markers. Use of the 12 high-resolution SSR markers revealed the exact same genetic profile between the mother tree (donor) and micropropagated plants, suggesting the genetic fidelity of our micropropagation protocol. The same protocol was also used successfully in propagating a “Golden Rain Tree” although response of explant and efficiency of propagation was much lower. This protocol will be useful for germplasm preservation/large scale production of true-to-type clones of desirable genotypes.

Dexamethasone influences FGF-induced responses in lens epithelial explants and promotes the posterior capsule coverage that is a feature of glucocorticoid-induced cataract

Aberrant spreading of lens epithelial cells along the posterior capsule is the basis for development of glucocorticoid (GC)-induced cataract; the resulting foci of nucleated cells at the posterior pole causing disruptions to normal lens cellular architecture. In this study, rat lens epithelial explants were used to assess the effects of dexamethasone (DEX), a widely used synthetic GC, on FGF2-induced lens cell proliferation and elongation as well as the ability of lens cells to spread and cover the posterior capsule. In the presence of FGF2, DEX significantly promoted lens cell proliferation after 48 h. Cell coverage of the posterior capsule was also enhanced during 5 days culture. In contrast, cell elongation was retarded by the inclusion of DEX. In the absence of FGF2, DEX had no marked effects on any of these cellular processes. Thus, in the presence of FGF2, DEX promoted cell proliferation and posterior capsule coverage but inhibited cell elongation. These results provide insights into the molecular mechanism underlying GC-induced cataract in humans.

Response of root explants to in vitro cultivation of marketable garlic cultivars

Garlic cultivars are sexually sterile under standard growth conditions, with direct implications for commercial production costs as well as breeding programs. Garlic is propagated commercially via bulblets, which facilitates disease transmission and virus load accumulation over vegetative generations. Tissue culture produces virus-free clones that are more productive, while keeping the desired traits of the cultivar. Consequently, this technique allows studies of garlic genetics as well as guarantees genetic conservation of varieties. We aimed at analyzing the in vitro regeneration of eight marketable cultivars of garlic using root segments as explants. For each genotype, bulblet-derived explants were isolated and introduced into MS medium supplemented with 2,4-D and 2-iP. Calli were transferred to MS medium supplemented with 8.8 mM BAP and 0.1 mM NAA (regeneration medium A), or with 4.6 mM kinetin alone (regeneration medium B). The calli were then evaluated for regeneration frequency after sixty days of in vitro cultivation. The noble cultivar 'Jonas' presented the highest rates of plant regeneration among the cultivars tested. The medium A, which contained auxin and cytokinin, induced the highest regeneration rates of all cultivars. The process described herein is simple, reproducible and can potentially be used as a tool in molecular breeding strategies for other marketable cultivars and genotypes of garlic.

Regeneration and Agrobacterium-mediated transformation of three economically important strawberry cultivars Kurdistan, Camarosa and Paros

Genetic transformation studies were carried out to standardize a protocol for Agrobacterium-mediated genetic transformation of three economically important strawberry (Fragaria x ananassa Duch) cultivars Kurdistan’, Camarosa’, and ‘Paros’. Shoot regeneration frequency 72, 65 and 30% was obtained on MS (1) basal medium supplemented with 2% glucose and 4 mg/l TDZ for Camarosa, Kurdistan and Paros, respectively. To optimize the concentration of kanamycin for these cultivars, we observed the responses of leaf explants to different concentration of kanamycin (0-100 mg/l) in the media. Our results showed that 75 mg/l was an effective concentration of kanamycin. For genetic transformation, Agrobacterium tumefaciens strain C58C1RifR (pGV2260) harbouring the binary vector pTJK136 containing uidA gene with intron along with kanamycin resistance gene (npt-II) was used. After five days pre-incubation and 72 h co-cultivation, transformed cells (explants) were able to grow on the selective regeneration medium containing 75 mg/l kanamycin and 500 mg/l cefotaxime, while, control explants failed to grow. The regenerated putative transgenic shoots were analyzed by histochemical GUS assay and PCR analysis. Transformation efficiency based on inoculated explants number was 2, 1 and 3% for ‘, Camarosa’, ‘Paros ’, and ‘Kurdistan’, respectively. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of these cultivars with important agronomic genes.

In vitro rooting and biochemical parameters in the cherry rootstocks CAB-6P and Gisela 6 using L-methionine

In the present study, the effects of indole-3-butyric acid (IBA) alone and in combination with L-methionine were investigated on the morphogenic and biochemical responses in the shoot tip explants of the cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (Prunus cerasus × Prunus canescens). The best results for roots, regarding their number and the fresh and dry weight, were obtained with 2 mg L^{-1} IBA in both rootstocks. Maximum rooting percentage was with the combination of 2 mg L^{-1} IBA and 0.5 mg L^{-1} L-methionine. Root length was greatest in the control plants. L-methionine had an inhibitory effect on the leaf chlorophyll content of the CAB-6P rootstock and no effect in Gisela 6. L-methionine with the lowest IBA concentration (0.5 mg L^{-1}) exhibited elevated levels of proline in the roots of the CAB-6P rootstock and depleted levels in Gisela 6. For the Gisela 6 rootstock, the leaf carbohydrate concentration was highest with 2 mg L^{-1} of IBA, whereas in the roots it was highest in the control. For the CAB-6P rootstock, the leaf carbohydrate concentration was not influenced by IBA with L-methionine, whereas the highest carbohydrate concentration in the roots was with the combinations of 0.5 mg L^{-1} IBA and 1 and 2 mg L^{-1} L-methionine. Taking into account the different parameters examined, it appears that chlorophyll and carbohydrates are the most accurate biochemical markers of the rooting process in both rootstocks. The increased levels of proline in the roots of CAB-6P with the treatments of 0.5 mg L^{-1} IBA combined with 1 and 2 mg of L^{-1} L-methionine, as opposed to IBA alone, show osmotic or oxidative stress. In the Gisela 6 explants, the decline in proline levels in the roots indicates a mechanism of osmoregulation and osmotic adjustment, which seems to be localized only in the roots and not in the leaves.

An Uncommon Plant Growth Regulator, Diethyl Aminoethyl Hexanoate, Is Highly Effective in Tissue Cultures of the Important Medicinal Plant Purple Coneflower (Echinacea purpureaL.)

We investigated the effects of various concentrations of diethyl aminoethyl hexanoate (DA-6) on the regeneration and growth of adventitious buds in in vitro purple coneflower cultures. Among the 3 types of explants tested, leaf explants required higher concentrations of DA-6 than petiole and root explants in order to obtain high regeneration rates, while root explants required the lowest concentration of DA-6. Additionally, explants with higher ploidy levels were more sensitive to the addition of DA-6, while explants with lower ploidy levels required higher concentrations of DA-6 to achieve its maximal regeneration rate. Interestingly, the application of a concentration that was conducive to the regeneration of explants with lower ploidy levels was inhibitory to the regeneration of explants with higher ploidy levels. Moreover, during the growth of regenerated buds, DA-6 application significantly improved plant height and weight, root weight, root thickness, root number, primary root length, total root length, and root/top ratio. Differences in the responses of explants to supplementation with DA-6 were also observed among explants with different ploidy levels, with buds having lower ploidy levels responding to lower concentrations of DA-6. Taken together, the results of the present experiments showed that proper application of DA-6 could increase in vitro culture efficiency in purple coneflower.

Potential role of cytokinin–auxin synergism, antioxidant enzymes activities and appraisal of genetic stability in Dianthus caryophyllus L.—an important cut flower crop

The regeneration potential, antioxidative enzyme activities, and genetic stability among micropropagated plantlets of Dianthus caryophyllus L. were evaluated. Multiple adventitious shoots were induced from leaf explants on Murashige and Skoog medium incorporated with various combinations and concentrations of plant growth regulators (PGRs). The highest leaf explant response (90%), number of shoots per explant (15.30 ± 1.19), and shoot length (6.75 ± 0.63 cm) was recorded in response to a combination of 2.5 μM 6-benzyladenine and 0.5 μM α-naphthaleneacetic acid (NAA) after 8 wks culture. Subsequent subculturing for five passages, on a medium with the same composition of PGRs, induced the highest shoot number (42.50 ± 1.44), with an average shoot length of 8.06 cm after the fourth subculture. Different concentrations of indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with a pulse treatment of IBA (100 μM) applied to the basal end of the microshoot for 30 min, followed by transfer to plastic cups containing soilrite, and eventually established in natural soil with an 85% survival rate. The determination of activities of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase) revealed involvement of these enzymes in shoot differentiation and development. All of these activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. Intersimple sequence repeat DNA analysis was carried out using five primers. The amplification products were monomorphic in micropropagated plants, similar to those of the mother plant. No polymorphisms were detected revealing the genetic integrity of the micropropagated plants.

Basophils are recruited to inflamed lungs and exacerbate memory Th2 responses in mice and humans

Although the contribution of basophils as inducers or amplifiers of Th2 responses is still debated, prolonged basophil/CD4 T cell interactions were observed in lungs but not lymph nodes (LNs) of parasite-infected mice. However, the impact of basophils on the function of tissue CD4 effector T cells remains unknown. Basophils were purified from the lungs of ovalbumin (OVA)-sensitized and OVA-challenged (OVA-immunized) mice or human peripheral blood for in vivo and in vitro functional studies. Pulmonary basophils were adoptively transferred to OVA-sensitized hosts to assess airway inflammation in bronchoalveolar lavage fluid (BALF) and Th2 responses in lung explants and draining LNs. Basophils were co-cultured with effector T cells or Ag-specific naïve T cells alone or in combination with dendritic cells (DCs); IL-4 production was determined by flow cytometry and ELISA. Basophils accumulated in lungs of OVA-immunized mice. Adoptive transfer of basophils to OVA-sensitized hosts enhanced lung IL-4 and IL-13 release while co-administration of OVA further aggravated airway inflammation and Th2 responses in LNs. Mechanistic in vitro studies revealed that pulmonary basophils interacted with lung CD4 effectors, in the absence of DCs, to increase T cell survival and Th2 cytokine expression at the single cell level but amplified OVA-loaded DC-driven Th2 differentiation. Finally, human basophils augmented in vitro IL-4 expression in effector memory CD4 T cells that include CRTH2(+) cells through IL-4 and TCR-independent pathways. Basophils may worsen Th2 inflammatory disorders through direct interactions with pathogenic CD4 T cells as well as by enhancing DC-induced Th2 cell development.

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes

Context: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size.Objectives: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model.Materials and methods: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7–615 nm; size 2: 616 nm–2.39 µm; size 3: 2.4–10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics.Results: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways.Discussion and conclusions: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0μM benzyl adenine (BA) and tender coconut water (10%) showed significantly high (P < 0.05) explant response (67.0%), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6% rooting on MS medium containing 15μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1%) ex vitro, by 10mM indole-3-butyric acid (IBA) for 30s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

Multiple shoot induction from axillary bud cultures of the medicinal orchid, Dendrobium longicornu.

Dendrobium longicornu, commonly known as the 'Long-horned Dendrobium', is an endangered and medicinally important epiphytic orchid. Over-exploitation and habitat destruction seriously threaten this orchid in Northeast India. Our objective was to develop an efficient protocol for the mass propagation of D. longicornu using axillary bud segments. Axillary buds cultured in Murashige and Skoog semi-solid medium supplemented with α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) readily developed into plantlets. These formed either directly from shoot buds or from intermediary protocorm-like bodies (PLBs). The maximum explant response (86.6 %) was obtained in medium supplemented with NAA at 30 µM, while the maximum number of shoots (4.42) and maximum bud-forming capacity (3.51) were observed in medium containing 15 µM BAP and 5 µM NAA in combination. Protocorm-like bodies were obtained when the medium contained 2,4-D. The maximum number of explants forming PLBs (41.48 %) was obtained in medium containing 15 µM BAP and 15 µM 2,4-D. Well-developed plantlets obtained after 20-25 weeks of culture were acclimatized and eventually transferred to the greenhouse. Over 60 % of these survived to form plants ∼3-4 cm tall after 90 days in glasshouse conditions using a substrate of crushed brick and charcoal, shredded bark and moss. The method described can readily be used for the rapid and large-scale regeneration of D. longicornu. Its commercial adoption would reduce the collection of this medicinally important and increasingly rare orchid from the wild.

In vitro organogenesis of rangpur lime

Rangpur lime (Citrus limonia Osbeck) in vitro organogenesis was studied based on explant type and cytokinin culture media supplementation. Four explants types collected from epicotyl or hypocotyl regions of in vitro germinated seedlings were evaluated. The epicotyl-derived explants consisted of epicotyl segments and the hypocotyl-derived explants consisted of the entire hypocotyl segment, the hypocotyl segment attached to a cotyledon fragment, and the hypocotyl segment divided longitudinally. The explants were cultured on EME culture medium supplemented with benzylaminopurine (0, 0.5, 1.0, or 1.5 mg L-1). The evaluation was performed after 6 weeks. Best results considering both the explant responsiveness and number of shoots developed per explants were obtained when epicotyl segments-derived explants were evaluated. Considering the explant responsiveness of hypocotyl segments-derived explants no difference was detected between the entire hypocotyl segment and the hypocotyl segment attached to a cotyledon fragment. Moreover, the percentage of responsive explants decreased when hypocotyl segments divided longitudinally were tested. No difference was detected for the number of shoots developed per explant considering the three types of hypocotyl-derived explants. Culture media supplementation with BAP was not essential for Rangpur lime in vitro organogenesis. However, adventitious shoot development was stimulated in concentrations between 0.5 - 1.0 mg L-1.

Long days promote somatic embryogenesis in spinach

As has been previously shown, the genotype at the individual plant level has a strong effect on the regeneration capacity of spinach, and this effect quite often masks the impact of other factors, including the light environment. Therefore, apical sections of the lateral roots of the same seedling (line) were exposed in parallel to both long day (LD, 16-h photoperiod) and short day (SD, 8-h photoperiod) conditions to assess the effect of photoperiod on somatic embryo (SE) regeneration. The root sections in most lines responded with significantly higher frequencies of regeneration and mean SE numbers with LD compared to roots of the same origin with SD, while 7 of 40 lines were only able to regenerate when exposed to LD. Moreover, in all lines cultured with SD, regeneration was delayed by approximately 4 weeks. Obtained SEs were further grown and multiplied, providing material for the evaluation of the effect of light intensity on regeneration capacity. Maximal regeneration capacity was recorded at 100μmolm−2s−1 in all lines tested. The present research confirmed the previously observed strong effect of genotype on regeneration capacity and undoubtedly showed a significantly better response in explants exposed to LD than to SD. These results could contribute to better understanding the process of SE induction in spinach and further improve the efficiency of this process.