Induction and identification of tetraploid <Emphasis Type="Italic">Hedychium coronarium</Emphasis> through thin cell layer culture

We describe an encapsulation–dehydration procedure with prefreezing steps for the cryopreservation of rhizome bud explants of Asparagus officinalis L. cv. Morado de Huétor. With this procedure, survival of Rhizome buds was at least 84 and 42% developed to complete plantlets at 8 weeks. Flow cytometry and EST-SSR molecular markers were used to assess genetic stability of the regenerated material. Effects of preculture time in a medium rich in sucrose and prefreezing treatments (0 °C or/and ??20 °C) on plant recovery were evaluated. Rhizome Buds of the “Morado de Huétor” landrace were incubated in preculture medium (MS?+?0.3 M sucrose) for 48 h, encapsulated in alginate beads and desiccated until a water content of 35%, prefrozen for one hour at 0 °C plus one hour at ? 20 °C, followed by cryopreservation in liquid nitrogen, and then were rewarmed and recovered in ARBM medium for 6 weeks and finally incubated in ARBM-0 for 4 weeks. Analyses of ploidy and molecular stability of plantlets recovered from cryopreserved rhizome buds of two selected genotypes showed no differences compared with the mother plants. Cryopreservation of RB explants of A. officinalis with this Encapsulation–Dehydration procedure will be useful in long-term preservation programs.     

Micropropagation of <Emphasis Type="Italic">Asparagus macrorrhizus</Emphasis>, a Spanish endemic species in extreme extinction risk

Asparagus macrorrhizus: is a new species, which has been recently described. It is limited to the area surrounding the “Mar Menor” lagoon, in Murcia (Spain), and is the only “Critically Endangered” species of the genus Asparagus. Despite being protected, the number of plants has decreased in the last years due to the urbanization of its natural habitat. This species is a valuable genetic resource for asparagus breeding because of its special characteristics. So, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The micropropagation protocol from asparagus rhizome buds previously developed by our research group has been adapted for A. macrorrhizus. Rhizome buds of A. macrorrhizus were extracted, disinfected, and then cultured on Asparagus Rhizome Bud Medium (ARBM) consisting of MS medium supplemented with 0.3 mg l^??1 NAA, 0.1 mg l^??1 KIN, 2 mg l^??1 ancymidol and 6% sucrose. A percentage of 69.7?±?8.0% of the rhizome buds developed shoots, but only 17.4?±?7.9% of them rooted. To increase this low rooting rate, the shoots were cultured on Macrorrhizus Rooting Media (MRM) supplemented with three different concentrations of IBA. The highest rooting rate (55.0?±?7.9%) was reached when shoots were incubated in MRM-2 consisting of MS medium supplemented with 2 mg l^??1 IBA and 4% sucrose. The acclimatization rate of the micropropagated plantlets was 90%. The method developed in this study allows the micropropagation of A. macrorrhizus, offering a new option to preserve this almost extinct species.     

Origin of tetraploid cultivated asparagus landraces inferred from nuclear ribosomal DNA internal transcribed spacers’ polymorphisms

The genus Asparagus includes a group of wild species that are closely related to the cultivated Asparagus officinalis (2n = 2× = 20). The narrow genetic background present in the asparagus cultivars shows the importance of asparagus landraces and the wild related species. The study of both genetic resources becomes necessary to facilitate their effective use in the breeding programmes. ‘Morado de Huetor’ (MH) and ‘Violetto d’Albenga’ (VA) are tetraploid asparagus landraces (2n = 4× = 40) cultivated in Spain and Italy, respectively, and whose origin remains unknown. To discover the origin of these landraces, a phylogenetic study was conducted based on restriction fragment length polymorphism (RFLP) of nuclear ribosomal DNA (nrDNA). The sequence of the two internal transcribed spacers (ITS) flanking the nrDNA5.8S gene (ITS1‐5.8S‐ITS2) were analysed for RFLP in 11 populations including both landraces (MH and VA), A. officinalis (wild and cultivated) and a group of closely related wild species (Asparagus maritimus, Asparagus prostratus, Asparagus pseudoscaber and Asparagus tenuifolius) with a European distribution. Restriction fragment patterns of both cultivated asparagus (2×) and two populations of A. maritimus (6×) from the Adriatic Sea area were present in the MH landrace. However, VA showed a similar pattern to A. officinalis. This study revealed that MH seems to be a hybrid between A. officinalis and A. maritimus that may have occurred in the Adriatic Sea region where hybridisations between cultivated diploid and wild species may have taken place. The origin of another tetraploid landrace (VA) might have had a similar origin but followed a different evolutionary path. Therefore, these landraces constitute a valuable genetic resource that could be used to enlarge the genetic background of modern cultivars. The ploidy levels of the populations employed in this study were analysed and levels not described previously were detected: A. maritimus (12×), A. tenuifolius (6×) and A. pseudoscaber (2×).     

The Effect of Nutritional Factors and Plant Growth Regulators on Micropropagation and Production of Phenolic Acids and Saponins from Plantlets and Adventitious Root Cultures of Eryngium maritimum L.

Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L^?1 benzyladenine (BA) and 0.1 mg L^?1 indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L^?1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.     

Synergic Effect of Exercise and Lipoic Acid on Protection Against Kainic Acid Induced Seizure Activity and Oxidative Stress in Mice

Anti-convulsant effects of physical exercise and lipoic acid (LA), also referred to as thioctic acid with antioxidant activity, were investigated using chemical induced seizure model. We investigated the synergic effect of physical exercise and LA on kainic acid-induced seizure activity caused by oxidative stress. After 8 weeks of swimming training, body weight decreased and endurance capacity increased significantly compared to sedentary mice. Kainic acid (30 mg/kg, i.p.) evoked seizure activity 5 min after injection, and seizure activity peaked approximately 80 min after kainic acid treatment. Median seizure activity score in KA only treated group was 4.55 (range 0.5–5), 3.45 for “LA + KA” group (range 0.5–4.3), 3.12 for “EX + KA” group (range 0.05–3.4, p < 0.05 vs. “KA only” group), 2.13 for “EX + LA + KA” group (range 0.5–3.0, p < 0.05 vs. “EX + KA” group). Also, there was a synergic cooperation of exercise and LA in lowering the mortality in kainic acid treated mice (χ² = 5.45, p = 0.031; “EX + KA” group vs. “LA + EX + KA” group). In addition, the synergic effect of exercise and LA was found in PGx activity compared to separated treatment (“LA + EX + KA”: 37.3 ± 1.36; p < 0.05 vs. “LA + KA” and “EX + KA” group). These results indicate that physical exercise along with LA could be a more efficient method for modulating seizure activity and oxidative stress.     

Regeneration of Transgenic Plants from Electroporated Protoplasts of Asparagus officinalis L

An effective method for consistent regeneration of transgenic asparagus (Asparagus officinalis L) plants from electroporated protoplasts is described. Transgenic plants containing β-glucuronidase (GUS) and neomycin-phosphotransferase (NPT II) genes were obtained by electroporating callus-derived protoplasts of Asparagus officinalis L. Embryogenic callus tissue and plants from four kanamycin resistant lines expressed P-glucuronidase activity, as revealed by histological staining. The amplification of genomic DNA by polymerase chain reaction revealed the presence of both GUS and NPT II genes in transformed callus tissue and plants. Southern hybridization confirmed the integration of these genes into the asparagus genome.     

Micropropagation of Laburnum anagyroides Medic. through axillary shoot regeneration

An in vitro propagation system based on the proliferation of axillary buds has been developed for Laburnum anagyroides. Culture initiation was influenced by explanting season, with the maximum response obtained from explants harvested in spring and autumn while the lowest response was noted in summer explants. The best shoot induction was observed on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzylaminopurine (BAP). The basal media, type of cytokinin, and explant type were the most important factors affecting shoot multiplication of L. anagyroides. Medium comprised of ½MS salts was found to be more efficient for axillary shoot multiplication compared to full-strength MS or Wood Plant Medium (WPM) when using identical growth regulators. Shoot tip explants were more responsive than nodal segments of microshoots for micropropagation. In vitro-derived shoots, >10 mm in length, were successfully rooted in medium containing ¼MS salts and 2.68 μM α-naphtalene acetic acid (NAA). In vitro-regenerated plantlets were adapted to ex vitro conditions and transferred to a greenhouse. This is the first report for successful in vitro propagation of L. anagyroides from bud explants of mature trees.     

Miscanthus: Genetic Diversity and Genotype Identification Using ISSR and RAPD Markers

Due to the limited number of molecular studies focused on European gene pool investigation, it is necessary to perform plant material recognition. Eighteen accessions of three Miscanthus species, namely, M. × giganteus, M. sinensis, M. sacchariflorus were evaluated with the use of molecular marker systems such as: inter simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPD), and by estimation of ploidy level based on flow cytometry. As a result, only one ISSR primer (ISSR1) and three RAPD primers (RAPD1, RAPD2, RAPD4) were required to identify all genotypes. Moreover, the use of the above mentioned molecular markers enable the proper species recognition of the interspecific hybrid M. × giganteus “Floridulus,” which has been previously mislabeled as M. floridulus. The highest genetic similarity coefficient (0.94) was observed between M. × giganteus clones, which indicates that the genetic diversity within this species was very low. Whereas M. sinensis genotypes represented a relatively wide diversity with similarity coefficient of 0.58. Cluster analysis using UPGMA grouped the 18 accessions in three clusters according to species affiliation including relabeled M. × giganteus “Floridulus,” which proved to be closely related to M.  × giganteus. Similar groupings were evident in the PCoA analysis.     

Effect of temperature on development and reproduction of Proprioseiopsis asetus (Acari: Phytoseiidae) fed on asparagus thrips,Thrips tabaci

Thrips tabaci Lindeman (Thysanoptera: Thripidae) is one of the most important pests of asparagus in China. In this study the effects of five constant temperatures (15, 20, 25, 30 and 35 °C) on the growth, survivorship and reproduction of Proprioseiopsis asetus (Chant) (Acari: Phytoseiidae) fed on T. tabaci was examined under laboratory conditions. Development time of immatures decreased with increasing temperature. The lower egg-to-adult developmental threshold (T ₀) and thermal constant (K) of P. asetus were estimated at 15.2 °C and 75.8 degree days by means of a linear model. Fertilized females fed on T. tabaci produced offspring of both sexes, whereas the offspring sex ratio [♀/(♀ + ♂)] of P. asetus at 20–35 °C was female-biased (0.68–0.78) and not significantly influenced by temperature. Survivorship during immature development was significantly influenced by temperature, and was especially low at 15 °C. Pre- and post-oviposition periods of fertilized females shortened with the increase in temperature. The longest oviposition period was 20.4 days, at 25 °C, whereas at 15 °C the mites did not reproduce. Maximum average life time fecundity and mean daily fecundity was recorded at 25 and 35 °C, respectively; the intrinsic rate of increase ranged from 0.05 (20 °C) to 0.17 (35 °C). The results indicate the capability of P. asetus to develop and reproduce at a broad range of temperatures, especially above 25 °C, which can be used for better management of T. tabaci in asparagus.     

An innovative approach to <Emphasis Type="Italic">ex vitro</Emphasis> rooting and acclimatization of <Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch. microshoots using а biogenic silica- and green-tea-catechin-based mechanocomposite

A new approach for rapid ex vitro rooting and acclimatization of Fragaria × ananassa micropropagated plantlets of two cultivars (“Alpha” and “Festivalnaya”) has been developed using a mechanocomposite based on biogenic silica and green-tea catechins. Two different mechanocomposite treatments were studied: dipping the cut ends of microshoots in the mechanocomposite powder (the dry dip method) and single watering with solutions at concentrations of 0.3, 1.0, and 3.0 g L^?1. These variants were compared with pulse treatment of microplants with 30 mg L^?1 indole-3-acetic acid (IAA) for 4 h and a control group of microshoots that were moistened with hormone-free ¼-strength MS medium. The frequencies of ex vitro rooting at the end of the acclimatization period (30 d) varied from 24.8 to 99.7%. The dry dip treatment was best (rooting frequency about 100%) with up to 7.15?±?0.54-cm root length, and 6.10?±?0.31 roots per plantlet. Moreover, this study showed that the growth-stimulating effect of this mechanocomposite treatment on root formation resulted in increased rosette height, leaf number, leaf area, and dry weight of aerial parts. Histological analysis of the leaf blades revealed decreased mesophyll thickness of microshoots treated with the mechanocomposite (up to 88.77?±?2.95 vs. 111.51?±?3.56 μm for the control). Morphometric analysis of scanning electron microscopy data showed that mechanocomposite treatments led to increased stomata density and stomata length. These structural changes led to normalization of the water regime and indicated successful acclimatization. The combination of ex vitro rooting and acclimatization reduced the procedure time by 4 wk, and may be used for commercial strawberry micropropagation.     

Identifying candidate genes for discrimination of ulcerative colitis and Crohn’s disease

In this study we aimed to screen effective biomarkers for differential diagnosis of ulcerative colitis (UC) and Crohn’s disease (CD). By using the gene expression profile dataset GSE24287 including 47 ileal CD, 27 UC and 25 non-inflammatory bowel diseases control downloaded from Gene Expression Omnibus database, we identified the differentially expressed genes (DEGs) between UC patients and controls as well as between CD patients and controls (|log₂FC(fold change)| > 1 and p < 0.05). Then Gene Ontology (GO) functional enrichment analyses were performed for these DEGs in two groups, followed by the construction of weight PPI (protein–protein interaction) networks. Subnets enriched for the PPIs and differentially expressed genes were constructed based on the weight PPI networks. The overlapping genes between the genes in the top 10 subnets with smallest p value and the DEGs were selected as the candidate genes of disease. A total of 75 DEGs were identified in UC group and 87 ones in CD group. There were 69 and 57 specific DEGs in CD group and UC group, respectively. The DEGs in CD group were mainly enriched in “inflammatory response” and “defense response”, while the most significantly enriched GO terms in UC group were “anion transport” and “chemotaxis”. FOS and SOCS3 were identified as candidate genes for CD and other three genes HELB, ZBTB16 and FAM107A were candidate genes for UC. In conclusion, there were distinct genetic alterations between UC and CD. The candidate genes identified in current study may be used as biomarkers for differential diagnosis of CD and UC.     

Effects of metabolic pathway precursors and polydimethylsiloxane (PDMS) on poly-(gamma)-glutamic acid production by Bacillus subtilis BL53

The aims of this study were to evaluate the effects of the addition of metabolic precursors and polydimethylsiloxane (PDMS) as an oxygen carrier to cultures of Bacillus subtilis BL53 during the production of γ-PGA. Kinetics analyses of cultivations of different media showed that B. subtilis BL53 is an exogenous glutamic acid-dependent strain. When the metabolic pathway precursors of γ-PGA synthesis, l-glutamine and a-ketoglutaric acid, were added to the culture medium, production of the biopolymer was increased by 20 % considering the medium without these precursors. The addition of 10 % of the oxygen carrier PDMS to cultures caused a two-fold increase in the volumetric oxygen mass transfer coefficient (k_La), improving γ-PGA production and productivity. Finally, bioreactor cultures of B. subtilis BL53 adopting the combination of optimized medium E, added of glutamine, α-ketoglutaric acid, and PDMS, showed a productivity of 1 g L^?1 h^?1 of g-PGA after only 24 h of cultivation. Results of this study suggest that the use of metabolic pathway precursors glutamine and a-ketolgutaric acid, combined with the addition of PDMS as an oxygen carrier in bioreactors, can improve γ-PGA production and productivity by Bacillus strains .     

Obolinga (Mimosaceae)—its natural history

Obolinga zanonii is endemic to the mountain chain of the Sierra de Bahoruco (Dominican Republic)-Massif de la Selle (Haiti) in southern Hispaniola. Its habitat is the humid broadleaf forest (“cloud forest”) at approximately 1500 m. Little is known about the dispersal of the seeds, but many fall and germinate below the parent tree. Germination in a nursery occurs in about 30 days.     

Understanding sampling and taxonomic biases recorded by citizen scientists

Projects involving citizen scientists have greatly increased over the last decade and understanding errors associated with such projects has been identified as an important step. NatureWatch NZ is a biodiversity recording system accessible to members of the public. The “NZ wasps, ants, bees and parasitoids (Hymenoptera) project” was initiated within NatureWatch NZ in December 2012, and comparisons were analysed between these records and the known Hymenoptera fauna of the New Zealand region. Over the course of 1 year 25 members contributed 360 records from 186 taxa, including the discovery of several introduced species new to New Zealand. There was a strong geographical bias to the records, with the majority being based around the major cities. Aculeates (stinging wasps) were significantly over-represented in the NatureWatch records. Only half (55 %) of taxa were identified to species level, with a further 28 % at genus level, and 17 % identified above genus level (family, order). Furthermore, the majority (65 %) of taxa were recorded only once, and only a few taxa were recorded >5 times (top records were “Ichneumonidae”, “Hymenoptera”, Anthidium manicatum, and Apis mellifera). It is probable that these same biases also exist for many other taxonomic groups in projects operated by citizen scientists lacking set protocols. Caution should be exercised on the subsequent use, compilation, and analysis of citizen science, especially without prior examination of records and potential biases.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Chromosome-scale haplotype-phased genome assemblies of the male and female lines of wild asparagus (Asparagus kiusianus), a dioecious plant species

Asparagus kiusianus is a disease-resistant dioecious plant species and a wild relative of garden asparagus (Asparagus officinalis). To enhance A. kiusianus genomic resources, advance plant science, and facilitate asparagus breeding, we determined the genome sequences of the male and female lines of A. kiusianus. Genome sequence reads obtained with a linked-read technology were assembled into four haplotype-phased contig sequences (∼1.6 Gb each) for the male and female lines. The contig sequences were aligned onto the chromosome sequences of garden asparagus to construct pseudomolecule sequences. Approximately 55,000 potential protein-encoding genes were predicted in each genome assembly, and ∼70% of the genome sequence was annotated as repetitive. Comparative analysis of the genomes of the two species revealed structural and sequence variants between the two species as well as between the male and female lines of each species. Genes with high sequence similarity with the male-specific sex determinant gene in A. officinalis, MSE1/AoMYB35/AspTDF1, were presented in the genomes of the male line but absent from the female genome assemblies. Overall, the genome sequence assemblies, gene sequences, and structural and sequence variants determined in this study will reveal the genetic mechanisms underlying sexual differentiation in plants, and will accelerate disease-resistance breeding in garden asparagus.     

Root-Associated Endophytic Bacterial Community Composition of Asparagus officinalis of Three Different Varieties

Asparagus (Asparagus officinalis L) is an economically important crop, rich in nutrients, and is also conducive to solving ecological and environmental problems. Plants may acquire benefits from root-associated endophytic bacteria. However, the composition of the endophytic bacterial community associated with the roots of asparagus is poorly elucidated. In this study, the nine root samples of asparagus from three different varieties including Asparagus officinalis var. Grande (GLD), A. officinalis var. Jinglvlu3 (JL3) and A. officinalis var. Jingzilu2 (JZL) were investigated by high-throughput sequencing technology of the 16S rDNA V5-V7 hypervariable region of endophytic bacteria. A total of 16 phyla, 29 classes, 90 orders, 171 families, and 312 genera were identified. Endophytic bacteria diversity and bacteria structure was different among the three varieties and was influenced by rhizosphere soil properties and varieties. In the GLD variety, the main phyla were Proteobacteria, Actinobacteria, and Firmicutes. The main phylum in JL3 and JZL varieties was Proteobacteria. The observations showed that GLD had the highest diversity of endophytes as indicated by the Shannon index (GLD > JZL > JL3). The order of the endophytes richness was GLD > JL3 > JZL. The PCA and PCoA analysis revealed the microbial communities were different between three different asparagus varieties, and the microbial composition of GLD and JZL was more similar. This report provides an important reference for the study of endophytic microorganisms of asparagus. Supplementary informationThe online version contains supplementary material available at (10.1007/s12088-021-00926-6) contains supplementary material, which is available to authorized users.     

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between “ON” and “OFF” by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA–RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at “ON” state and that of pepc and ecaA genes were controlled at the “OFF” state in the lag phase and switched to the “OFF” and “ON” state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g^?1 and 3.25 g L^?1 h^?1, respectively, much higher than those using the strains without harboring the riboregulator switch system.