Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species.

Medicinal plants are integral to traditional medicine systems world-wide, being pivotal for human health. Harvesting plant material from natural environments, however, has led to species scarcity, prompting action to develop cultivation solutions that also aid conservation efforts. Biotechnological tools, specifically plant tissue culture and genetic transformation, offer solutions for sustainable, large-scale production and enhanced yield of valuable biomolecules. While these techniques are instrumental to the development of the medicinal plant industry, the challenge of inherent regeneration recalcitrance in some species to in vitro cultivation hampers these efforts. This review examines the strategies for overcoming recalcitrance in medicinal plants using a holistic approach, emphasising the meticulous choice of explants, e.g. embryonic/meristematic tissues; plant growth regulators, e.g. synthetic cytokinins; and use of novel regeneration-enabling methods to deliver morphogenic genes e.g. GRF/GIF chimeras and nanoparticles, which have been shown to contribute to overcoming recalcitrance barriers in agriculture crops. Furthermore, it highlights the benefit of cost-effective genomic technologies that enable precise genome editing and the value of integrating data-driven models to address genotype-specific challenges in medicinal plant research. These advances mark a progressive step towards a future where medicinal plant cultivation is not only more efficient and predictable but also inherently sustainable, ensuring the continued availability and exploitation of these important plants for current and future generations.

Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa.

Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose-response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses.

Green Synthesized Zinc Oxide Nanoparticles as Feed Additives to Improve Growth, Biochemical, and Hematological Parameters in Freshwater Fish Labeo rohita.

Zinc deficiency in aquatic animals affects the biological processes and physiological functions. Thus, the supplement of ZnONPs can be used as an alternative method to overcome zinc deficiency. Nanoparticles have the potential to enhance the growth and health of the fish. The main aim of this study is to evaluate the growth efficacy of ZnONP-supplemented diet with fingerlings of Labeo rohita. The green synthesized ZnONPs were characterized by ultraviolet-visible (UV-Vis) spectroscopy, Fourier transformer infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Different concentrations of ZnONPs (5, 7.5, and 10 mg/kg) were administered in the basal diet of freshwater fish Labeo rohita for 45 days to observe the growth and metabolic functions of the body. However, the fish fed with 10 mg/kg ZnONP-supplemented diet shows that the growth performance was highly increased followed by 7.5 mg/kg and 5 mg/kg ZnONPs when compared with the control. The biochemical, hematological, and digestive enzyme activities were also significantly increased with different concentrations of ZnONPs. The effects of zinc oxide nanoparticles show the higher improvement of growth and metabolic functions in Labeo rohita. These results suggest that the nanotechnology could apply for feed formulation technology and pave the way for the dietary supplementation of zinc oxide nanoparticles as safe ingredients for aquatic animals to overcome the zinc deficiency.

KNS4/UPEX1: A Type II Arabinogalactan ß-(1,3)-Galactosyltransferase Required for Pollen Exine Development.

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific ß-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess ß-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.

DEFECTIVE KERNEL1 (DEK1) Regulates Cell Walls in the Leaf Epidermis.

The plant epidermis is crucial to survival, regulating interactions with the environment and controlling plant growth. The phytocalpain DEFECTIVE KERNEL1 (DEK1) is a master regulator of epidermal differentiation and maintenance, acting upstream of epidermis-specific transcription factors, and is required for correct cell adhesion. It is currently unclear how changes in DEK1 lead to cellular defects in the epidermis and the pathways through which DEK1 acts. We have combined growth kinematic studies, cell wall analysis, and transcriptional analysis of genes downstream of DEK1 to determine the cause of phenotypic changes observed in DEK1-modulated lines of Arabidopsis (Arabidopsis thaliana). We reveal a novel role for DEK1 in the regulation of leaf epidermal cell wall structure. Lines with altered DEK1 activity have epidermis-specific changes in the thickness and polysaccharide composition of cell walls that likely underlie the loss of adhesion between epidermal cells in plants with reduced levels of DEK1 and changes in leaf shape and size in plants constitutively overexpressing the active CALPAIN domain of DEK1. Calpain-overexpressing plants also have increased levels of cellulose and pectins in epidermal cell walls, and this is correlated with the expression of several cell wall-related genes, linking transcriptional regulation downstream of DEK1 with cellular effects. These findings significantly advance our understanding of the role of the epidermal cell walls in growth regulation and establish a new role for DEK1 in pathways regulating epidermal cell wall deposition and remodeling.

Molecular modeling on streptolysin-O of multidrug resistant Streptococcus pyogenes and computer aided screening and in vitro assay for novel herbal inhibitors.

Streptococcus pyogenes is a notorious pathogenic bacterium which causes various human diseases ranging from localized infections to life threatening invasive diseases. Streptolysin-O (SLO), pore-forming thiol-activated cytolysin, is the major virulent factor for streptococcal infections. Present therapies against streptococcal infections are limited as most of the strains have developed multi-drug resistance to present generation of drugs. Hence, there is a need for alternative therapeutic substances. Structure based virtual screening is a novel platform to select lead molecules with better pharmacokinetic properties. The 3D structure of SLO (not available in native form), essential for such studies, was computationally generated and this homology model was used as probable drug target. Based on literature survey, several phytoligands from 25 medicinal plants were selected. Out of these, leads from 11 plants showed better pharmacokinetic properties. The best lead molecules were screened based on computer aided drug likeness and pharmacokinetic predictions. The inhibitory properties of selected herbal leads against SLO were studied by molecular docking. An in vitro assay was further carried out and variations observed were found to be significant (p<0.05). Antibiotic sensitivity testing was also performed with the clinical strain of Streptococcus pyogenes with conventional drugs. The clinical strain showed multi-drug resistance to conventional drugs. Our study revealed that numerous phytoligands have better inhibitory properties towards the toxin. We noticed that incorporation of selected herbal extracts in blood agar medium showed significant reduction in hemolysis (MIC 300µl/plate), indicating inhibition of SLO. Furthermore, the butanol extracts of selected herbal preparation based on computer aided screening showed significant inhibitory properties at 250 mcg/disc concentration. We also noticed that selected herbal formulations have better antimicrobial properties at MIC range of 300- 400µl. Hence, our study suggests that these herbal extracts have better inhibitory properties against the toxin as well as drug resistant Streptococcus pyogenes.

Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.

Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I. Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences.

A case-control study of risk factors for lung cancer in Mumbai, India.

In the year 2010, it is estimated that nearly 1.35 million new cases and 1.18 million deaths with lung cancer occurred. In India, among males, lung cancer rates vary across the country which has encouraged us to conduct a case-control study to study the risk factors. The present unmatched hospital-based case-control study conducted at Tata Memorial Hospital included subjects registered between the years 1997-99. There were 408 lung 'cancer cases' and 1383 'normal controls'. Data on age, tobacco habits, occupational history, dietary factors, tea, coffee were collected by the social investigators. Univariate and regression analysis were applied for obtaining the odds ratio for risk factors. In the study, cigarette smoking (OR=5.2) and bidi smoking (OR=8.3), as well as alcohol consumption (OR=1.8), demonstrated dose-response relationships with lung cancer risk. Among the dietary items, only red-meat consumption showed 2.2-fold significant excess risk. Consumption of milk showed a 60% reduction in risk; while coffee showed a 2-fold excess risk for lung cancer. In addition, exposure to use of pesticides showed a 2.5-fold significant excess risk for lung cancer.