<i>Agrobacterium rhizogenes</i> vs auxinic induction for <i>in vitro</i> rhizogenesis of <i>Prosopis chilensis</i> and <i>Nothofagus alpina</i>

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg.L^-1 benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg.L^-1 of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg.L^-1 IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg.L^-1 IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.     

Optimization of the <i>in-situ</i> growth conditions based on a novel photo-microcalorimeter for the sustainable cultivation of photosynthetic microorganisms

Despite the great potential of photosynthetic microbes in the production of renewable fuels, value-adding chemicals, and water treatment, etc., commercial utilization of them is significantly hindered by the lack of techniques to accurately monitor the thermodynamic and kinetic characteristics of the In-situ growth of microbes under controlled light illumination for optimal cultivation. Herein, we demonstrated that a newly developed highly sensitive photo-microcalorimetric system successfully captured the impacts of the light wavelength and strength on the thermodynamic and kinetic parameters of the In-situ growth of Rhodopseudomonas palustris, a representative photosynthetic microorganism. To our best knowledge, this is the first time that highly precise microcalorimetry is employed to monitor exam the in-situ growth of photosynthetic microorganisms under controllable photo illumination. We envision this technique can help for the optimization of the growth conditions of photosynthetic microorganisms for scale-up applications.     

<i>Claroideoglomus etunicatum</i> improved the growth and saline– alkaline tolerance of <i>Potentilla anserina</i> by altering physiological and biochemical properties

To investigate the effects of arbuscular mycorrhizal (AM) fungi on the growth and saline–alkaline tolerance of Potentilla anserina L., the seedlings were inoculated with Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schüßler in pot cultivation. After 90 days of culture, saline–alkaline stress was induced with NaCl and NaHCO₃ solution according to the main salt components in saline–alkaline soils. Based on the physiological response of P. anserina to the stress in the preliminary experiment, the solution concentrations of 0 mmol/L, 75 mmol/L, 150 mmol/L, 225 mmol/L and 300 mmol/L were treated with stress for 10 days, respectively. The mycorrhizal colonization rate, mycorrhizal dependence, chlorophyll content, malondialdehyde content, antioxidant enzyme activities, osmoregulation substances content and water status were measured. The results showed that with the increase of NaCl and NaHCO₃ stress concentration, mycorrhizal colonization rate, colonization intensity, arbuscular abundance and vesicle abundance decreased, and reached the lowest value at 300 mmol/L. Strong mycorrhizal dependence was observed after the symbiosis with AM fungus, and the dependence was higher under NaHCO₃ treatment. Under NaCl and NaHCO₃ stress, inoculation with AM fungus could increase chlorophyll content, decrease malondialdehyde content, increase activities of superoxide dismutase, peroxidase and catalase, increase contents of proline, soluble sugar and soluble protein, increase tissue relative water content and decrease water saturation deficit. It was concluded that salt–alkali stress inhibited the colonization of AM fungus, but the mycorrhiza still played a positive role in maintaining the normal growth of plants under salt–alkali stress.     

Brief Note : Improved <i>in vitro</i> embryo development of stenospermic grape by putrescine

The goal of this study was to determine the effect of putrescine, added to the culture medium, on the in vitro development of stenospermic grape (Vitis vinifera L) embryos. The cross breedings of Perlón x G.C88552 and Perlón x Argentina were used. 0 (control), 2 and 4 mM of putrescine were added to the immature seed’s culture medium. In Perlón x Argentina, 2mM of putrescine statistically increased the percentage of total embryos, direct germination, polyembryos and normal plants. In Perlón x G.C88552, only 2 mM of putrescine increased all the variables considered, eventually tripling the percentage of normal plants obtained. The results suggest that the endogenous concentration of putrescine may be a growth limiting factor. Adding putrescine to the culture medium of immature grape seeds is a legitimate resource to significantly increase the results of this technique.     

A 66-kDa protein of bovine hypophyseal <i>Pars tuberalis</i> induces luteinizing hormone release from rat <i>Pars distalis</i>

In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 μg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 μg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis.     

<i>In vitro</i> propagation of <i>Opuntia ellisiana</i> Griff. and acclimatization to field conditions

The genus Opuntia is a valuable forage resource in arid and semiarid lands during periods of drought and shortage of herbaceous plants. However, absolute minimum temperatures in the plains of Mendoza represent a limiting factor to cultivate several species.
Opuntia ellisiana is a cold hardy species, so the goals of this study were to massively propagate it using in vitro culture techniques, and then to acclimatize plantlets obtained to field conditions.
Different sterilization protocols were tested. Areoles were isolated in laminar airflow cabinet, and cultured on Murashige-Skoog medium, supplemented with sucrose and different BAP and IBA combinations. Explants were grown at 27±2ºC, under a 16-h photoperiod. The shoots produced were used in the rooting assay using different auxin combinations. In the most efficient growth treatment, plantlets reached 100% shooting after 35 days of culture, and a mean length of 10.2 mm after 49 days of culture. A 100% rooted plantlets was obtained on a medium containing 5 mg L^-1 IBA, after 12 days of culture. Acclimatization was achieved under greenhouse conditions, showing 100% plantlet survival.
This study suggests that O. ellisiana can be successfully micropropagated by areoles, and easily acclimatizated to field conditions.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Impact of pituitary FSH purification on <i>in vitro</i> early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol^® and Folltropin^®) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol^® (50 ng/mL) or Folltropin^® (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol^® maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin^® at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin^® than Stimufol^®. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol^® was better to preserve follicular morphology while Folltropin^® was more efficient to promote follicular growth.     

Micropropagation of <i>Ilex dumosa</i> (Aquifoliaceae) from nodal segments in a tissue culture system

Micropropagation of Ilex dumosa var. dumosa R. (“yerba señorita”) from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in ¹/₄ strength Murashige and Skoog medium (¹/₄ MS) plus 30 gr·L^-1 sucrose and supplemented with 4.4 µM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr·L^-1 sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in ¹/₄ MS (30 gr·L^-1 sucrose, 0.25 % Phytagel^®) with 7.3 µM IBA and 2) 21 days in the same medium without IBA and 20 µM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.     

Organogenesis and plant regeneration of <i>Arachis villosa</i> Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: α-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 μM thidiazuron and 4.44 μM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 μM α-naphthalenacetic acid, 13.95 μM kinetin and 13.32 μM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 μM α-naphthalenacetic acid.     

Micropropagation of <i>Prosopis chilensis</i> (Mol.) Stuntz from young and mature plants

Prosopis chilensis (Mol.) Stuntz (Algarrobo de Chile) is an important native tree species that can be grown in arid and semiarid regions for wood and forage production and environmental protection. Developing a simple and reliable in vitro protocol for cloning it would enable to improve it genetically. Explants of P.chilensis were taken from 4 months-old plants grown in the greenhouse or from adult trees grown in a natural environment. Nodal segments 1 – 2 cm long containing an axillary bud were selected from elongating shoots. These cuttings were aseptically cultured on two agar-solid basal media, MS or BTMm, and treated with 0.05 mg L^-1 BA and 3 mg L^-1 of either IAA, IBA or NAA. Sucrose (3% w/v) was used as carbon source. The percentage of sprouted cuttings and whole plant regeneration as well as its shoot and root length were recorded. Number, length and dry weight of shoots and roots were also measured. Rooting was successful with cuttings taken from young or adult plants, but explants from young plants showed a better response. Culturing in BTMm resulted in significantly greater shoot and root biomass than culturing in MS. Moreover, this response was higher in young explants when IBA was used as growth regulator. This paper reports a simple and effective method to micropropagate P. chilensis from young and adult plants.     

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in <i>Escherichia coli</i> to promote L-tryptophan production

In this study, phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas (EMP) pathway and pentose phosphate (PP) pathway in Escherichia coli, thus increasing the L-tryptophan production. Firstly, the effects of disrupting EMP pathway on L-tryptophan production were studied, and the results indicated that the strain with deletion of phosphofructokinase A (i.e., E. coli JW-5 ΔpfkA) produced 23.4 ± 2.1 g/L of L-tryptophan production. However, deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth, especially deletion of glucosephosphate isomerase. Next, the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd) and thus increasing the L-tryptophan production (i.e., 26.5 ± 3.2 g/L vs. 21.7 ± 1.3 g/L) without obviously changing the cell growth (i.e., 0.41 h⁻¹ vs. 0.44 h⁻¹) as compared with the original strain JW-5. Finally, the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated. It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd (i.e., E. coli JW-5 zwf243 gnd361 ΔpfkA) produced 31.9 ± 2.7 g/L of L-tryptophan, which was 47.0% higher than that of strain JW-5. In addition, the glucose consumption rate of strain JW-5 zwf243 gnd361 ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5. The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.     

Characterization of multidrug-resistant <i>Klebsiella pneumoniae</i> isolated from the Chinese cobra <i>Naja atra</i> in a Beijing suburb

The emergence and spread of antibiotic resistance genes among Bacteria are a serious threat to global health. Their occurrence in animals which are in contact with humans is also important. The Chinese cobra (Naja atra, Elapidae), though a highly venomous species, is appreciated as food and as a source of materials used in traditional Chinese medicine. We are here reporting the isolation of multidrug-resistant Klebsiella pneumoniae (Enterobacteriaceae) from the lung of Naja atra, obtained from a snake farm in a Beijing suburb. Our study analyzed, using gene sequencing, the occurrence of antibiotic resistance genes (ARGs) in three K. pneumoniae isolates from two snakes. In addition, bacterial clones were identified by biochemical tests and phylogenetic analysis. Tests of antimicrobial susceptibility showed that all K. pneumoniae isolates were resistant to a host of antibiotics (piperacillin, cefazolin, gentamicin, tetracycline, doxycyclin, ciprofloxacin, levofloxacin, lomefloxacin, ofloxacin, norfloxacin, nalidixic acid, chloramphenicol, nitrofurantoin, sulfamethoxazole, and sulfamethoxazole/trimethoprim) but were susceptible to cefotaxime, cefixime, aztreonam, bramycin, amikacin, kanamycin, netilmicin, and streptomycin. Eighteen ARGs were detected in total DNA extracted from the isolates. Results showed three quinolone resistance genes (oqxA, oqxB, qnrB), the gyrA gene that confers resistance to beta-lactam antibiotics, and the emerging aac(3)-II gene that confers resistance to aminoglycosides. K. pneumoniae is an important opportunistic human pathogen and the emergence of multidrug-resistant K. pneumoniae in N. atra suggests the increasing risk of pathogen transmission between humans, livestock, and wildlife. Given the close association between foodborne pathogenic microorganisms and humans, it is key factor to identify these antibiotic resistance genes profile thereby minimize the risk of K. pneumoniae transmission.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Brief Note : Micropropagation of <i>Glandularia perakii</i> Cov. et Schn. (Verbenaceae), a native species with ornamental potential

Glandularia perakii is a perennial species with beautiful violet flowers that grows in the stony soil of Mendocine pedemont. A plentiful and prolonged flowering confers it an important ornamental potential. In this paper, a method of propagation of G. perakii from nodal segments is reported. Proliferating microshoot cultures were obtained by placing nodal segment on Murashige and Skoog medium (MS) supplemented with 20 g.L^-1 of sucrose without growth regulators. In this medium multiplication rate after 20 days was 7.9. Rooted plants were acclimatized successfully .     

Identification of <i>PtGai</i> (a DELLA protein) in trifoliate orange and expression patterns in response to drought stress

Gibberellins (GAs) are an important hormone in regulating plant growth and development, and DELLA protein is an essential negative regulator of GA signal transduction. The aim of the study was to clone a GA-inhibiting protein DELLA from trifoliate orange (Poncirus trifoliata L. Raf.) and to analyze the bioinformations and expression patterns of the protein gene in tissues and in response to drought stress. A DELLA protein was isolated from trifoliate orange and named as PtGai (Genebank number: MZ170959). The PtGai protein had 1731 bp open reading frames, along with 576 amino acid codes, and also grouped with sweet orange (XM_006430552.4). The PtGai protein sequence was 65% homology with the sequences of DELLA proteins in other plant families. PtGai protein existed in the nucleus based on the prediction of subcellular localization. PtGai protein could be expressed in roots, stems, and leaves, along with the highest expression in stems. PtGai was upregulated by drought stress in leaves and roots, along with the decrease of root total GA concentration and the inhibition of shoot and root biomass production. It indicated the characteristics of PtGai protein and the roles of PtGai in GA synthesis and plant growth.     

Induction of adaptive response <i>in utero </i>by ionizing radiation: A radiation quality dependent phenomenon

Investigation on possible induction of adaptive response (AR) by high-liner energy transfer (LET) particle radiation for protection against low-LET photon radiation-induced detrimental effects has not yet been performed in utero. This study verified if an AR could be induced by high-LET particle radiation from accelerated heavy ions against low-LET X-ray radiation-induced detrimental effects on fetal mice. Total body irradiation of pregnant C57BL/6J mice were performed by delivering a priming dose ranging from 10 mGy to 320 mGy of particle radiation on gestation day 11 followed one day later by a challenge dose at 3500 mGy from X-ray radiation. The monoenergetic beams of carbon, silicon and iron with the LET values of about 15, 55, and 200 KeV/μm, respectively, were examined. Significant suppression by the priming radiation of the detrimental effects (fetal death, malformation, or low body weight) was used as the endpoints for judgment of a successful AR induction on gestation day 18. Existence of AR was not observed. On the other hand, the priming dose of high-LET particle radiation, in some cases, even increased the detrimental effects induced by the challenge dose from low-LET X-ray radiation. Although existence of AR induced by high-LET radiation in cultured mammalian cells in vitro and in certain tissues of laboratory mice in vivo was demonstrated, the present study did not suggest that low dose of high-LET particle radiation could induce an AR in fetal mice in utero under the setup of our experimental system.