Guinea Worm Disease Presenting as a Subcutaneous Calcification.

A female patient in her early 50s presented with a complaint of multiple right thigh swellings for eight years which on evaluation were found to be guinea worms. The patient had no risk factors as she was living in a rural area of Maharashtra. We performed radiological investigations which only revealed calcifications in subcutaneous planes. On exploring the excised specimen post-surgery there was a worm-like appearance which on histopathological study confirmed guinea worm disease. We highlight this unusual presentation of guinea worm disease so that surgeons can treat the condition effectively.

Adenomyoepithelioma: A Case Report of a Rare Breast Lump.

Adenomyoepithelioma (AME) of the breast is a rare tumor characterized by biphasic differentiation into luminal and myoepithelial cells, with various histological patterns observed. This case report details a 35-year-old female with a progressively enlarging breast lump diagnosed initially as a fibroadenoma through ultrasonography (USG) and fine-needle aspiration cytology (FNAC). The patient underwent successful excision of the lump under general anesthesia, with histopathological examination confirming a benign tumor comprising epithelial and myoepithelial cells. This case underscores the importance of comprehensive clinical assessment and accurate diagnostic techniques in managing breast lumps, emphasizing the need for timely intervention for favorable outcomes.

External Jugular Vein Slow-Flow Venous Malformation Presenting as a Swelling Over the Neck: A Report of a Rare Case.

Vascular malformations originating from the wall of the external jugular vein are exceedingly uncommon. We present a unique case of a venous malformation arising from the external jugular vein, successfully treated through surgical excision with no subsequent recurrence. This case highlights the importance of early diagnosis and timely intervention in managing such rare clinical entities without any resulting morbidity.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

MAIN CONCLUSION: The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

Genome-wide identification and gene expression analysis of GHMP kinase gene family in banana cv. Rasthali.

BACKGROUND: The GHMP kinase gene family encompasses ATP-dependent kinases, significantly involved in the biosynthesis of isoprenes, amino acids, and metabolism of carbohydrates. Banana is a staple tropical crop that is globally consumed but known for high sensitivity to salt, cold, and drought stresses. The GHMP kinases are known to play a significant role during abiotic stresses in plants. The present study emphasizes the role of GHMP kinases in various abiotic stress conditions in banana. METHODS AND RESULTS: We identified 12 GHMP kinase (MaGHMP kinase) genes in the banana genome database and witnessed the presence of the conserved Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala domain in their protein sequences. All genes were found to be involved in ATP-binding and carried kinase activity confronting their biological roles in the isoprene (27%) and amino acid (20%) biosyntheses. The expression analysis of genes during cold, drought, and salt stress conditions in tissue culture grown banana cultivar Rasthali plants showed a significant involvement of MaGHMP kinase genes in these stress conditions. The highest expression of MaGHMP kinase3 (8.5 fold) was noted during cold stress, while MaGHMP kinase1 (25 fold and 40.01 fold) showed maximum expression during drought and salt stress conditions in leaf tissue of Rasthali. CONCLUSION: Our findings suggested that MaGHMP kinase1 (MaHSK) and MaGHMP kinase3 (MaGlcAK) could be considered promising candidates for thwarting the abiotic stresses in banana.

Anti-amyloidogenic amphipathic arginine-dehydrophenylalanine spheres capped selenium nanoparticles as potent therapeutic moieties for Alzheimer's disease.

Aggregation of both amyloid beta (Aß) peptide and hyperphosphorylated tau proteins is the major pathological hallmark of Alzheimer's disease (AD). Moieties that carry anti-amyloidogenic potency against both of the aggregating entities are considered to be promising drug candidatures for the disease. In the current work, we have synthesized amphipathic dipeptide vesicle-templated selenium nanoparticles (RΔF-SeNPs) as potential entities to combat AD. We have investigated and established their anti-amyloidogenic activity against different peptide-based amyloid models, such as the reductionist model based on the dipeptide phenylalanine-phenylalanine (FF) derived from Aß; a model based on the hexapeptide Ac-PHF6 (306VQIVYK311) derived from tau protein; and the full-length Aß42 polypeptide-based model. We also evaluated the neuroprotective characteristics of RΔF-SeNPs against FF, Ac-PHF6, and Aß42 fibril-induced toxicity in neuroblastoma, SH-SY5Y cells. RΔF-SeNPs further exhibited neuroprotective effects in streptozotocin (STZ) treated neuronal (N2a) cells carrying AD-like features. In addition, studies conducted in an intra-cerebroventricular STZ-instigated rat model of dementia revealed that RΔF-SeNP-treated animals showed improved cognitive activity and reduced Aß42 aggregate burden in brain tissues as compared with the STZ-treated group. Moreover, in vivo brain distribution studies conducted in animal models additionally demonstrated the brain-homing ability of RΔF-SeNPs. All together, these studies supported the potency of RΔF-SeNPs as efficient and propitious disease-modifying therapeutic agents for combating AD.

Overexpression of banana GDP-L-galactose phosphorylase (GGP) modulates the biosynthesis of ascorbic acid in Arabidopsis thaliana.

l-Ascorbic acid (AsA) is a potent antioxidant and essential micronutrient for the growth and development of plants and animals. AsA is predominantly synthesized by the Smirnoff-Wheeler (SW) pathway in plants where the GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting step. In the present study, AsA was estimated in twelve banana cultivars, where Nendran carried the highest (17.2 mg/100 g) amount of AsA in ripe fruit pulp. Five GGP genes were identified from the banana genome database, and they were located at chromosome 6 (4 MaGGPs) and chromosome 10 (1 MaGGP). Based on in-silico analysis, three potential MaGGP genes were isolated from the cultivar Nendran and subsequently overexpressed in Arabidopsis thaliana. Significant enhancement in AsA (1.52 to 2.20 fold) level was noted in the leaves of all three MaGGPs overexpressing lines as compared to non-transformed control plants. Among all, MaGGP2 emerged as a potential candidate for AsA biofortification in plants. Further, the complementation assay of Arabidopsis thaliana vtc-5-1 and vtc-5-2 mutants with MaGGP genes overcome the AsA deficiency that showed improved plant growth as compared to non-transformed control plants. This study lends strong affirmation towards development of AsA biofortified plants, particularly the staples that sustain the personages in developing countries.

Wheat derived glucuronokinase as a potential target for regulating ascorbic acid and phytic acid content with increased root length under drought and ABA stresses in Arabidopsis thaliana.

Glucuronokinase (GlcAK) converts glucuronic acid into glucuronic acid-1-phosphate, which is then converted into UDP-glucuronic acid (UDP-GlcA) via myo-inositol oxygenase (MIOX) pathway. UDP-GlcA acts as a precursor in the synthesis of nucleotide-sugar moieties forming cell wall biomass. GlcAK being present at the bifurcation point between UDP-GlcA and ascorbic acid (AsA) biosyntheses, makes it necessary to study its role in plants. In this study, the three homoeologs of GlcAK gene from hexaploid wheat were overexpressed in Arabidopsis thaliana. The GlcAK overexpressing transgenic lines showed decreased contents of AsA and phytic acid (PA) as compared to control plants. Root length and seed germination analyses under abiotic stress (drought and abscisic acid) conditions revealed enhanced root length in transgenic lines as compared to control plants. These results indicate that the MIOX pathway might be contributing towards AsA biosynthesis as evident by the decreased AsA content in the GlcAK overexpressing transgenic Arabidopsis thaliana plants. Findings of the present study will enhance the understanding of the involvement of GlcAK gene in MIOX pathway and subsequent physiological effects in plants.

A Perspective Review on Understanding Drought Stress Tolerance in Wild Banana Genetic Resources of Northeast India.

The enormous perennial monocotyledonous herb banana (Musa spp.), which includes dessert and cooking varieties, is found in more than 120 countries and is a member of the order Zingiberales and family Musaceae. The production of bananas requires a certain amount of precipitation throughout the year, and its scarcity reduces productivity in rain-fed banana-growing areas due to drought stress. To increase the tolerance of banana crops to drought stress, it is necessary to explore crop wild relatives (CWRs) of banana. Although molecular genetic pathways involved in drought stress tolerance of cultivated banana have been uncovered and understood with the introduction of high-throughput DNA sequencing technology, next-generation sequencing (NGS) techniques, and numerous "omics" tools, unfortunately, such approaches have not been thoroughly implemented to utilize the huge potential of wild genetic resources of banana. In India, the northeastern region has been reported to have the highest diversity and distribution of Musaceae, with more than 30 taxa, 19 of which are unique to the area, accounting for around 81% of all wild species. As a result, the area is regarded as one of the main locations of origin for the Musaceae family. The understanding of the response of the banana genotypes of northeastern India belonging to different genome groups to water deficit stress at the molecular level will be useful for developing and improving drought tolerance in commercial banana cultivars not only in India but also worldwide. Hence, in the present review, we discuss the studies conducted to observe the effect of drought stress on different banana species. Moreover, the article highlights the tools and techniques that have been used or that can be used for exploring and understanding the molecular basis of differentially regulated genes and their networks in different drought stress-tolerant banana genotypes of northeast India, especially wild types, for unraveling their potential novel traits and genes.

Opportunities and challenges with CRISPR-Cas mediated homologous recombination based precise editing in plants and animals.

KEY MESSAGE: We summarise recent advancements to achieve higher homologous recombination based gene targeting efficiency in different animals and plants. The genome editing has revolutionized the agriculture and human therapeutic sectors by its ability to create precise, stable and predictable mutations in the genome. It depends upon targeted double-strand breaks induction by the engineered endonucleases, which then gets repaired by highly conserved endogenous DNA repair mechanisms. The repairing could be done either through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The HDR-based editing can be applied for precise gene targeting such as insertion of a new gene, gene replacement and altering of the regulatory sequence of a gene to control the existing protein expression. However, HDR-mediated editing is considered challenging because of lower efficiency in higher eukaryotes, thus, preventing its widespread application. This article reviews the recent progress of HDR-mediated editing and discusses novel strategies such as cell cycle synchronization, modulation of DNA damage repair factors, engineering of Cas protein favoring HDR and CRISPR-Cas reagents delivery methods to improve efficiency for generating knock-in events in both plants and animals. Further, multiplexing of described methods may be promising towards achieving higher donor template-assisted homologous recombination efficiency at the target locus.

Genome-wide identification and expression profiling of WUSCHEL-related homeobox (WOX) genes confer their roles in somatic embryogenesis, growth and abiotic stresses in banana.

Plant-specific WUSCHEL-related homeobox (WOX) transcription factors are known to be involved in plant developmental processes, especially in embryogenesis. In this study, a total of thirteen WOX members were identified in the banana (Musa acuminata) genome (MaWOX) and characterized for in-silico analysis. Phylogenetic analysis revealed that these genes were divided into three clades (ancient, intermediate and modern) which reflected the evolutionary history of WOX families. Furthermore, modern clade members have shown higher variations in gene structural features and carried unique conserved motifs (motif 3 and motif 4) when compared to the members of other clades. The differential expression of all 13 MaWOX was observed in early (embryogenic cell suspension (ECS), multiplying ECS, germinating embryos, young leaflet and node of germinated plantlets) and late (unripe fruit peel and pulp, ripe fruit peel and pulp) developmental stages of banana cultivar Grand Naine. The maximum expression of MaWOX6 (18 fold) and MaWOX13 (120 fold) was found during somatic embryogenesis and in unripe fruit pulp, respectively. Moreover, numerous cis-elements responsive to drought, cold, ethylene, methyl jasmonate (MeJA), abscisic acid (ABA) and gibberellic acid (GA) were observed in all MaWOX promoter regions. The subsequent expression analysis under various abiotic stresses (cold, drought and salt) revealed maximum expression of the MaWOX3 (830 fold), MaWOX8a (30 fold) and MaWOX11b (105 fold) in salt stress. It gives evidence about their possible role in salt stress tolerance in banana. Hence, the present study provides precise information on the MaWOX gene family and their expression in various tissues and stressful environmental conditions that may help to develop climate-resilient banana plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03387-w.

Analysis of TCP Transcription Factors Revealed Potential Roles in Plant Growth and Fusarium oxysporum f.sp. cubense Resistance in Banana (cv. Rasthali).

The TCP transcription factor gene family is highly conserved among the plant species. It plays a major role in the regulation of flower symmetry, cell division, and development of leaf, fibre, and nodule in the plants by controlling the synthesis of various plant hormones. Banana is a major staple crop in the world. However, Fusarium oxysporum f. sp. cubense (Foc) infection is a major threat to banana production. The role of TCP gene family during the Foc infection is not explored till now. Herein, a total of 27 non-redundant TCP (MaTCP) gene sequences were retrieved from the banana genome and analysed for structural characteristics, phylogenetic correlation, subcellular, and chromosomal localizations. Phylogenetic analysis showed that the MaTCP proteins were highly conserved among different species and found to be the closest relative of the Oryza sativa and Zea mays. Promoter analysis of the TCP sequences showed that the cis-acting regulatory elements are associated with various stresses and environmental and hormonal signals. The higher transcript accumulation in developing tissues (fruit finger, leaves, and stem) than of mature tissues (peel and pulp) showed a significant role of MaTCP in banana (cv. Rasthali) growth and development. Further, higher expression of the certain MaTCPs in Foc race 1-infected root (MaTCP2, MaTCP4, MaTCP6) and leaf (MaTCP9 and MaTCP11) tissues of Rasthali indicated their promising role during Fusarium infection. This study will underpin the facet of TCP transcription factors on the development of biotic (Foc) stress resistance in banana.

Metabolic engineering in food crops to enhance ascorbic acid production: crop biofortification perspectives for human health.

Ascorbic acid (AsA) also known as vitamin C is considered as an essential micronutrient in the diet of humans. The human body is unable to synthesize AsA, thus solely dependent on exogenous sources to accomplish the nutritional requirement. AsA plays a crucial role in different physiological aspects of human health like bone formation, iron absorption, maintenance and development of connective tissues, conversion of cholesterol to bile acid and production of serotonin. It carries antioxidant properties and is involved in curing various clinical disorders such as scurvy, viral infection, neurodegenerative diseases, cardiovascular diseases, anemia, and diabetes. It also plays a significant role in COVID-19 prevention and recovery by improving the oxygen index and enhancing the production of natural killer cells and T-lymphocytes. In plants, AsA plays important role in floral induction, seed germination, senescence, ROS regulation and photosynthesis. AsA is an essential counterpart of the antioxidant system and helps to defend the plants against abiotic and biotic stresses. Surprisingly, the deficiencies of AsA are spreading in both developed and developing countries. The amount of AsA in the major food crops such as wheat, rice, maize, and other raw natural plant foods is inadequate to fulfill its dietary requirements. Hence, the biofortification of AsA in staple crops would be feasible and cost-effective means of delivering AsA to populations that may have limited access to diverse diets and other interventions. In this review, we endeavor to provide information on the role of AsA in plants and human health, and also perused various biotechnological and agronomical approaches for elevating AsA content in food crops.

On the Relationship Between Pain Variability and Relief in Randomized Clinical Trials.

Previous research reports suggest greater baseline variability is associated with greater pain relief in those who receive a placebo. However, studies that evidence this association do not control for confounding effects from regression to the mean and natural history. In this report, we analyzed data from two randomized clinical trials (Placebo I and Placebo II, total N = 139) while adjusting for the effects of natural history and regression to the mean via a no treatment group. Results agree between the two placebo groups in each study: both placebo groups showed negligible semi-partial correlations between baseline variability and adjusted response [r sp (CI95%) = 0.22 (0.03, 0.42) and 0 (-0.07, 0.07) for Placebo I and II, respectively]. The no treatment group in Placebo I showed a negative correlation [-0.22 (-0.43, -0.02)], but the no treatment and drug groups in Placebo II's correlations were negligible [-0.02 (-0.08, 0.02) and 0.00 (-0.10, 0.12) for the no treatment and drug groups, respectively]. When modeled as a linear covariate, baseline pain variability accounted for <1% of the variance in post-intervention pain across both studies. Even after adjusting for baseline pain and natural history, the inability of baseline pain variability to account for substantial variance in pain response highlights that previous results concerning pain variability and treatment response may be inconsistent. Indeed, the relationship appears to be neither consistently specific nor sensitive to improvements in the placebo group. More work is needed to understand and establish the prognostic value of baseline pain variability-especially its placebo specificity and generalizability across patient populations.

Transgene-free genome editing supports CCD4 role as a negative regulator of ß-carotene in banana.

This study aims to understand the regulatory mechanism of the ß-carotene homeostasis by establishing transgene-free genome editing in banana. Carotenoid cleavage dioxygenases (CCDs) belong to a miniature gene family having an imperative role in the intricated carotenoid metabolism in plants. Here, the expression pattern of multiple CCDs was correlated with the levels of carotenoid accumulation in two contrasting cultivars, viz., Nendran (high ß-carotene) and Rasthali (low ß-carotene). The higher expression of the RAS-CCD4 inversely correlated with ß-carotene accumulation in fruit-pulp of the Rasthali. The docking analysis followed enzyme assay of purified RAS-CCD4 suggested ß-carotene and 10-apo-ß-carotenal as its preferred substrates. Bacterial complementation assay affirmed RAS-CCD4 role in ß-carotene degradation and then overexpression of the RAS-CCD4 in the Arabidopsis thaliana further validated results in-vivo by the significant reduction in ß-carotene. Subsequently, CRISPR/Cas9 mediated editing of CCD4 was demonstrated in the protoplasts and embryogenic cell lines of Rasthali. The carotenoid profiling in stable mutant lines revealed higher fold ß-carotene accumulation in non-green tissue (roots) than in green tissue (leaf) compared with the unedited control plants. The differential expression of carotenoid pathway genes was correlated with the metabolites in the edited lines. The study suggests that carotenoid catabolism is regulated by the CCD4 in tissue and cultivar specific manners, and also demonstrated the use of the genome editing tool in developing transgene-free biofortified banana.

Comparative transcriptome analysis of unripe and ripe banana (cv. Nendran) unraveling genes involved in ripening and other related processes.

Banana is one of the most important fruit crops consumed globally owing to its high nutritional value. Previously, we demonstrated that the ripe pulp of the banana cultivar (cv.) Nendran (AAB) contained a high amount of pro-vitamin A carotenoids. However, the molecular factors involved in the ripening process in Nendran fruit are unexplored. Hence, we commenced a transcriptome study by using the Illumina HiSeq 2500 at two stages i.e. unripe and ripe fruit-pulp of Nendran. Overall, 3474 up and 4727 down-regulated genes were obtained. A large number of identified transcripts were related to genes involved in ripening, cell wall degradation and aroma formation. Gene ontology analysis highlighted differentially expressed genes that play a key role in various pathways. These pathways were mainly linked to cellular, molecular and biological processes. The present transcriptome study also reveals a crucial role of up-regulated carotenoid biosynthesis pathway genes namely, lycopene beta cyclase and geranylgeranyl pyrophosphate synthase at the ripening stage. Genes related to the ripening and other processes like aroma and flavor were highly expressed in the ripe pulp. Expression of numerous transcription factor family genes was also identified. This study lays a path towards understanding the ripening, carotenoid accumulation and other related processes in banana.

Carotenoid cleavage dioxygenases (HD-CCD1A and B) contribute as strong negative regulators of ß-carotene in Indian bread wheat (cv. HD2967).

Wheat (Triticum aestivum L.) is the most common cereal crop that is considered to be deficient in provitamin A carotenoids. Carotenoids are prone to degrade into apocarotenoids by the activity of carotenoid cleavage dioxygenases (CCDs). Hence, in this study, multiple CCDs were cloned from commercial Indian wheat cultivar HD2967 to understand their role in provitamin A carotenoids degradation. The homoeolog specific expression of HD-CCD1 and HD-CCD4 at different grain filling stages revealed the higher expression of transcripts arising from the A and B subgenomes of HD-CCD1. Furthermore, the grain development stages showed a strong negative correlation of HD-CCD1A (r = - 0.969) and B (r = - 0.970) homoeologs expression to that of ß-carotene accumulation. It suggested that they could be potentially involved in deciding the turn-over of ß-carotene in wheat grain. Three-dimensional (3D) structures for all six homoeologs of HD-CCD1 and HD-CCD4 were predicted using maize VP14 template to gain better insight into their molecular mechanism. Ramachandran plot assessment revealed that ~ 90% of residues are in the most favoured region. Docking studies with various carotenoid substrates revealed the higher affinity of HD-CCD1A and B for ß-carotene and ß-cryptoxanthin. Bacterial complementation analysis validated the functional role of all six homoeologs with HD-CCD1B showing the highest activity followed by HD-CCD1A for ß-carotene degradation. Results of this study provide valuable insights into the characteristics of HD-CCDs in wheat and thereby justifying them (HD-CCD1A and B) as the candidate genes for employing genome editing tools for developing ß-carotene enriched wheat grains. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02775-y.

What Is the Numerical Nature of Pain Relief?

Pain relief, or a decrease in self-reported pain intensity, is frequently the primary outcome of pain clinical trials. Investigators commonly report pain relief in one of two ways: using raw units (additive) or using percentage units (multiplicative). However, additive and multiplicative scales have different assumptions and are incompatible with one another. In this work, we describe the assumptions and corollaries of additive and multiplicative models of pain relief to illuminate the issue from statistical and clinical perspectives. First, we explain the math underlying each model and illustrate these points using simulations, for which readers are assumed to have an understanding of linear regression. Next, we connect this math to clinical interpretations, stressing the importance of statistical models that accurately represent the underlying data; for example, how using percent pain relief can mislead clinicians if the data are actually additive. These theoretical discussions are supported by empirical data from four longitudinal studies of patients with subacute and chronic pain. Finally, we discuss self-reported pain intensity as a measurement construct, including its philosophical limitations and how clinical pain differs from acute pain measured during psychophysics experiments. This work has broad implications for clinical pain research, ranging from statistical modeling of trial data to the use of minimal clinically important differences and patient-clinician communication.

Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant.

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2-25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l-1) + naphthalene acetic acid (0.5 mg l-1), gibberellic acid (1.0 mg l-1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l-1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana.

The production of wheat is severely affected by abiotic stresses such as cold, drought, salinity, and high temperature. Although constitutive promoters are frequently used to regulate the expression of alien genes, these may lead to undesirable side-effects in transgenic plants. Therefore, identification and characterization of an inducible promoter that can express transgene only when exposed to stresses are of great importance in the genetic engineering of crop plants. Previous studies have indicated the abiotic stress-responsive behavior of myo-inositol oxygenase (MIOX) gene in different plants. Here, we isolated the MIOX gene promoter from wheat (TaMIOX). The in-silico analysis revealed the presence of various abiotic stress-responsive cis-elements in the promoter region. The TaMIOX promoter was fused with the UidA reporter gene and transformed into Arabidopsis thaliana. The T3 single-copy homozygous lines were analyzed for GUS activity using histochemical and fluorometric assays. Transcript expression of TaMIOX::UidA was significantly up-regulated by heat (five fold), cold (seven fold), and drought (five fold) stresses as compared to transgenic plants grown without stress-induced conditions. The CaMV35S::UidA plants showed very high GUS activity even in normal conditions. In contrast, the TaMIOX::UidA plants showed prominent GUS activity only in stress treatments (cold, heat, and drought), which suggests the inducible behavior of the TaMIOX promoter. The substrate myo-inositol feeding assay of TaMIOX::UidA plants showed lesser GUS activity as compared to plants treated in abiotic stress conditions. Results support that the TaMIOX promoter could be used as a potential candidate for conditional expression of the transgene in abiotic stress conditions.