Genetic Modification Research Articles

Light-emitting plants development by inoculating of Vibrio campbellii RMT1 on the rhizospheric zone of Aglaonema cochinchinense.

The concept of utilizing light-emitting plants (LEPs) as an alternative to traditional electricity-based lighting has garnered interest. However, challenges persist due to the need for genetic modification or chemical infusion in current LEPs. To address this, researchers have investigated the interaction between plants and luminous bacteria, specifically Vibrio campbellii, which can efficiently be translocated into Aglaonema cochinchinense tissues through the roots to produce LEPs. This study concentrated on examining light intensity and enhancing luminescence by growing plants and spraying them with various media substances. The results indicated that V. campbellii successfully translocated into the plant tissue via the root system and accumulated a high number of bacteria in the stems, approximately 8.46 × 104CFU/g, resulting in a light-emitting intensity increase of 12.13-fold at 48h, and then decreased after 30h. Interestingly, luminescence stimulation by spraying the growth medium managed to induce the highest light emission, reaching 14.84-fold at 48h, though it had some negative effects on the plant. Conversely, spraying plants with CaCl2 on the leaves prolonged light emission for a longer duration (42h after spraying) and had a positive effect on plant health, it maintained ion homeostasis and reduced-MDA content. This study highlights the potential of using V. campbellii and CaCl2 spraying for the future development of practical light-emitting plants.

Current Perspectives and Progress in Preoperative Portal Vein Embolization with Stem Cell Augmentation (PVESA).

Portal vein embolization with stem cell augmentation (PVESA) is an emerging approach for enhancing the growth of the liver segment that will remain after surgery (i.e., future liver remnant, FLR) in patients with liver cancer. Conventional portal vein embolization (PVE) aims to induce preoperative FLR growth, but it has a risk of failure in patients with underlying liver dysfunction and comorbid illnesses. PVESA combines PVE with stem cell therapy to potentially improve FLR size and function more effectively and efficiently. Various types of stem cells can help improve liver growth by secreting paracrine signals for hepatocyte growth or by transforming into hepatocytes. Mesenchymal stem cells (MSCs), unrestricted somatic stem cells, and small hepatocyte-like progenitor cells have been used to augment liver growth in preclinical animal models, while clinical studies have demonstrated the benefit of CD133 + bone marrow-derived MSCs and hematopoietic stem cells. These investigations have shown that PVESA is generally safe and enhances liver growth after PVE. However, optimizing the selection, collection, and application of stem cells remains crucial to maximize benefits and minimize risks. Additionally, advanced stem cell technologies, such as priming, genetic modification, and extracellular vesicle-based therapy, that could further enhance efficacy outcomes should be evaluated. Despite its potential, PVESA requires more investigations, particularly mechanistic studies that involve orthotopic animal models of liver cancer with concomitant liver injury as well as larger human trials.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Phosphatidic acid produced by phospholipase Dα1 and Dδ is incorporated into the internal membranes but not involved in the gene expression of RD29A in the abscisic acid signaling network in Arabidopsis thaliana

Core protein components of the abscisic acid (ABA) signaling network, pyrabactin resistance (PYR), protein phosphatases 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) are involved in the regulation of stomatal closure and gene expression downstream responses in Arabidopsis thaliana. Phosphatidic acid (PA) produced by the phospholipases Dα1 and Dδ (PLDs) in the plasma membrane has been identified as a necessary molecule in ABA-inducible stomatal closure. On the other hand, the involvement of PA in ABA-inducible gene expression has been suggested but remains a question. In this study, the involvement of PA in the ABA-inducible gene expression was examined in the model plant Arabidopsis thaliana and the canonical RD29A ABA-inducible gene that possesses a single ABA–responsive element (ABRE) in the promoter. The promoter activity and accumulation of the RD29A mRNA during ABA exposure to the plants were analyzed under conditions in which the production of PA by PLDs is abrogated through chemical and genetic modification. Changes in the subcellular localization of PA during the signal transduction were analyzed with confocal microscopy. The results obtained in this study suggest that inhibition of PA production by the PLDs does not affect the promoter activity of RD29A. PA produced by the PLDs and exogenously added PA in the plasma membrane are effectively incorporated into internal membranes to transduce the signal. However, exogenously added PA induces stomatal closure but not RD29A expression. This is because PA produced by the PLDs most likely inhibits the activity of not all but only the selected PP2C family members, the negative regulators of the RD29A promoter. This finding underscores the necessity for experimental verifications to adapt previous knowledge into a signaling network model before its construction.

Metabolic Engineering of Corynebacterium glutamicum for Highly Efficient Production of Ectoine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

Posttranscriptional regulation of FAN1 by miR-124-3p at rs3512 underlies onset-delaying genetic modification in Huntington’s disease

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.

Chromosome Transplantation: Opportunities and Limitations

There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These “genomic mutations” include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of “synthetic” cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.

Promoting mitochondrial dynamics by inhibiting the PINK1/PRKN pathway to relieve diabetic nephropathy.

Diabetes is a metabolic disorder characterized by high blood glucose levels and is a leading cause of kidney disease. Diabetic nephropathy has been attributed to dysfunctional mitochondria. However, many questions remain about the exact mechanism. The structure, function, and molecular pathways between mammalian podocytes and Drosophila nephrocytes are highly conserved, therefore we used flies on a high-sucrose diet to model type 2 diabetic nephropathy. The nephrocytes of high-sucrose diet flies showed significant functional decline and decreased cell size, associated with a shortened lifespan. Structurally, the nephrocytes filtration structure known as the slit diaphragm was disorganized. At the cellular level, we found altered mitochondrial dynamics and dysfunction. Regulating mitochondrial dynamics by either genetic modification of the Pink1/Park (mammalian PINK1/PRKN) pathway or treatment with BGP-15, mitigated the mitochondrial defects and nephrocyte functional decline. These findings support a role for Pink1/Park-mediated mitophagy and associated control of mitochondrial dynamics, essential for function, in diabetic nephropathy; and demonstrate that targeting this pathway might provide therapeutic benefits in type 2 diabetic nephropathy.

Regulation of Molecular Biomarkers Associated with the Progression of Prostate Cancer

Androgen receptor signaling regulates the normal and pathological growth of the prostate. In particular, the growth and survival of prostate cancer cells is initially dependent on androgen receptor signaling. Exposure to androgen deprivation therapy leads to the development of castration-resistant prostate cancer. There is a multitude of molecular and cellular changes that occur in prostate tumor cells, including the expression of neuroendocrine features and various biomarkers, which promotes the switch of cancer cells to androgen-independent growth. These biomarkers include transcription factors (TP53, REST, BRN2, INSM1, c-Myc), signaling molecules (PTEN, Aurora kinases, retinoblastoma tumor suppressor, calcium-binding proteins), and receptors (glucocorticoid, androgen receptor-variant 7), among others. It is believed that genetic modifications, therapeutic treatments, and changes in the tumor microenvironment are contributing factors to the progression of prostate cancers with significant heterogeneity in their phenotypic characteristics. However, it is not well understood how these phenotypic characteristics and molecular modifications arise under specific treatment conditions. In this work, we summarize some of the most important molecular changes associated with the progression of prostate cancers and we describe some of the factors involved in these cellular processes.

Clinical studies with drugs and biologics aimed at slowing or reversing normal aging processes—emerging results and future perspectives

Five families of investigational products are in clinical investigation to slow or reverse normal aging processes [longevity candidates, mesenchymal stem cells, senolytics drugs, sirtuin activators, and nicotinamide adenine dinucleotide (NAD)+ precursors]. The longevity candidates, vitamin D and metformin, appear to significantly reduce all-cause mortality and prolong life expectancy. This should be confirmed by interventional studies. The mesenchymal stem cell family is the most advanced in clinical trial development [phase 2b randomized controlled trial (RCT)]. An allogeneic bone marrow stem cell preparation (Lomecel-B) reduced locomotor frailty in older people. The improvement in locomotion was modest. In the future, attempts could be made to improve potency through a precondition or genetic modification of naive bone marrow stem cells. Autologous adipose stem cell-assisted fat grafting increased graft survival, facial volume, and skin quality. The association of the senolytic drugs dasatinib and quercetin was well tolerated, with low brain penetration of dasatinib and undetectable levels of quercetin. The sirtuin-1 activator resveratrol (combined with physical exercise) improved physical function in older adults with physical limitations. The NAD+ precursor nicotinamide riboside improved physical exercise performance. In conclusion, Lomecel-B is the most advanced agent in clinical trial development for normal aging processes (phase 2b for locomotion frailty), followed by resveratrol and nicotinamide riboside.

Role of genetically modified organisms in food, crop production, their regulations, and controversy

Abstract Estimates suggest that over one-third of food is lost or wasted along the food supply chain, with significant environmental and economic effects. This is one of the primary problems with today’s food systems, along with decreased agricultural yield and food quality. Many people attribute the decline in output and the deterioration of food quality to a lack of technology and ignorance about modern technologies. As a result, scientists are working hard to find innovative solutions to these problems and create new food production methods to feed the world’s growing population. Increased agricultural yields and quality may be achieved in a variety of settings by employing genetically engineered techniques. Newer techniques and technologies involving genetic modification and recombinant (r) deoxyribonucleic acid could be very helpful in the pursuit of significant improvements in food production and food supply, and biotechnology is a key part of this. Recent progress in genetic engineering, where it came from, the current trend, and its potential future are all discussed in this overview. Genetically modified organisms (GMOs) are major aspects of the industrial food system, and most packaged foods contain GMOs ingredients that have been bred to be resistant to pesticides and herbicides. There are a number of factors that raise red flags concerning GMOs, including safety, consequences on the environment and present ineffective employment of pesticides have also been considered. Furthermore, GMOs regulation and controversy among different scientists has also been discussed. Many individuals are concerned about GMOs, yet they may not fully grasp the concerns at hand. Therefore, the level of GMO knowledge revealed by this review can be calculated.

Continuous Secretion of Human Epidermal Growth Factor Based on Escherichia coli Biofilm

Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing.

Plasmonic-Driven Regulation of Biomolecular Activity In Situ.

Selective and remote manipulation of activity for biomolecules, including protein, DNA, and lipids, is crucial to elucidate the molecular function and to develop biomedical applications. While advances in tool development, such as optogenetics, have significantly impacted these directions, the requirement for genetic modification significantly limits their therapeutic applications. Plasmonic nanoparticle heating has brought new opportunities to the field, as hot nanoparticles are unique point heat sources at the nanoscale. In this review, we summarize fundamental engineering problems such as plasmonic heating and the resulting biomolecular responses. We highlight the biological responses and applications of manipulating biomolecules and provide perspectives for future directions in the field.

Biotechnological tools for genetic improvement of Trichoderma

Trichoderma is a cosmopolitan fungus widely distributed around the world. The different uses of this beneficial fungus are varied in several industries, like agriculture, textile, and paper, including the pharmaceutical industry. The genus Trichoderma has other mechanisms of action, including the production of different enzymes and secreted secondary metabolites used in various industries. The genomes of different Trichoderma species have been sequenced to identify the mechanisms for producing several compounds. The advancement of multiple technologies has allowed the development of transformation tools for the genetic improvement of Trichoderma, thus increasing biomass, primary and secondary metabolites, and enzymes. Therefore, genetic modification aims to increase compound production in several Trichoderma strains. Characterization of Trichoderma through gene expression analysis is essential for biotechnology applications. It helps counteract one of the most challenging problems for agriculture today, including climate change and the appearance of pathogens that attack crops with high commercial and food demand. In conclusion, this review analyzes various strategies to improve Trichoderma strains genetically and their multiple applications in the agricultural, textile, paper, and pharmaceutical industries. As a recommendation for future studies with potential impact, the optimization of specific genetic modifications in Trichoderma strains is recommended to improve their adaptability and effectiveness in combating emerging challenges in agriculture, especially those linked to climate change. Investigating possible synergies between genetically modified Trichoderma strains and environmentally sustainable agricultural practices could contribute to developing solutions for crop protection and yield improvement.

The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer

ABSTRACT Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain’s unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens ’ performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Sustainable approaches for removing toxic heavy metal from contaminated water: A comprehensive review of bioremediation and biosorption techniques

In this comprehensive study, highlights emerging environmentally friendly methods to eliminating hazardous heavy metals from contaminated water, with an emphasis on bioremediation and biosorption. Breakthroughs, such as the combination of biological remediation and nanotechnology to improve the elimination of metals effectiveness and the use of genetically modified microbes for targeted pollutant breakdown. Developing biosorption materials made from agricultural waste and biochar, this indicates interesting areas for future research and emphasizes the necessity of sustainable practices in tackling heavy metal contamination in water systems. There seems to be a surge in enthusiasm for the utilization of biological remediation and biosorption methods as sustainable and viable options for eliminating heavy metals from contaminated water in the past couple of decades. The present review intends to offer an in-depth review of the latest understanding and advances in the discipline of biological remediation methods like bioaccumulation, biofiltration, bio-slurping, and bio-venting. Biosorption is specifically explained and includes waste biomass as biosorbent with the removal mechanisms and the hindrances caused in the process are detailed. Advances in biosorption like microbes as biosorbents and the mechanism involved in it. Additionally, novel enhancement techniques like immobilization, genetic modification, and ultrasound-assisted treatment in microbial sorbent are clarified. However, the review extended with analyzing the future advances in the overall biological methods and consequences of heavy metal pollution.

Safety evaluation of the food enzyme subtilisin from the genetically modified Bacillus licheniformis strain NZYM-CB.

The food enzyme subtilisin (EC 3.4.21.62) is produced with the genetically modified Bacillus licheniformis strain NZYM-CB by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in six food manufacturing processes. The dietary exposure to the food enzyme-TOS was estimated to be up to 0.722 mg TOS/kg body weight (bw) per day in European populations. The production strain of the food enzyme fulfils the requirements for the qualified presumption of safety approach to safety assessment. As no other concerns arising from the manufacturing process were identified, the Panel considered that toxicological tests were not required for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 20 matches were found, including two food allergens (melon and pomegranate). The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, particularly in individuals sensitised to melon and pomegranate, but would not exceed the risk from consumption of melon or pomegranate. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Biochemical Changes in Embryogenic and Nonembryonic Callus of Glycyrrhiza glabra L. during Somatic Embryogenesis

The continuous extraction of glycyrrhizin from Glycyrrhiza glabra L. has led to the critical endangerment of this therapeutic plant. Glycyrrhizin, a triterpenoid saponin, is a vital secondary metabolite renowned for its diverse pharmacological benefits, including anti-inflammatory, immunomodulatory, antiallergic, antiulcer properties, and even efficacy against HIV. However, the relentless harvesting of roots for high-value glycyrrhizin results in the obliteration of the entire plant, posing a severe threat to its existence and causing a detrimental impact on biodiversity. To counteract this challenge and conserve the species, sustainable approaches are imperative. One such approach involves the utilization of in vitro regeneration through somatic embryogenesis for clonal proliferation and genetic modification. This report presents findings on SE-associated proteins in Glycyrrhiza glabra L., accomplished through the implementation of the discontinuous gel electrophoresis technique. The study focused on a comparative isozyme analysis employing non-embryogenic and embryogenic calli of G. glabra L. Remarkably, these isozymes were exclusively identified and exhibited affiliations with a spectrum of cellular functions. A substantial portion of these proteins are engaged in a diverse range of metabolic and stress-related functions. This study utilized crude enzyme/total protein extracts as a basis to evaluate the genetic stability of in vitro cultivated plant tissue. Acid phosphatase, esterase, and peroxidase were selected as indicators for this assessment. Intriguingly, esterase and acid phosphatase exhibited significant polymorphism between the segregated embryogenic callus (EC) and non-embryogenic callus (NEC) derived from the same G. glabra L. genotype. Comparative analyses encompassing esterase, acid phosphatase, and peroxidase activities, alongside the expression profiles of these isozymes throughout somatic embryogenesis (SE), were conducted using polyacrylamide gel electrophoresis. Notably, the isoenzyme patterns of calluses were assessed. The activity of isozymes within an embryogenic line of callus surpassed that within a non-embryogenic counterpart. However, no conspicuous alterations in quality were observed between the non-embryogenic and embryogenic calluses.